Increase In Isc Research Articles

Adrenoceptor-mediated secretion across the rat colonic epithelium

Norepinephrine evoked a biphasic change in short-circuit current (Isc) across the proximal and distal colon of the rat. The (1) phase of the current response consisted of a transient increase, which was followed by a long-lasting decrease during the (2) phase. The (1) phase, which is assumed to represent Cl − secretion, was resistant against classical adrenoceptor antagonists, but was inhibited by the β 3-adrenoceptor antagonist 3-(2-ethylpenoxy)-1-[(1 S-1,2,3,4-tetrahydronaphth-1-ylaminol-(2 S)-propranol oxalate (SR 59230A) in the proximal colon and by the non-selective β-adrenoceptor antagonist bupranolol in both colonic segments. Vice versa, the increase in Isc was mimicked by the β 3-adrenoceptor agonist, ( R*, R*)-(±)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344). The (2) phase of the norepinephrine-induced Isc, which is assumed to represent K + secretion, was inhibited by yohimbine in the proximal colon, suggesting the mediation by α 2-adrenoceptors, whereas in the distal colon, both α- and β-adrenoceptors are involved, as shown by the sensitivity against, e.g. phentolamine and propranolol. These adrenoceptors seem to be located — at least in part — at extraepithelial sites because the (1) phase of the norepinephrine response was sensitive to indomethacin, and the (2) phase, both to indomethacin and tetrodotoxin.

Nucleotide-evoked ion transport and [Ca 2+] i changes in normal and hyperhidrotic human sweat gland cells

Apical and basolateral application of ATP and UTP evoked [Ca 2+] i and short circuit current (Isc) increases in normal and hyperhidrotic human eccrine sweat gland cells grown into functionally polarised epithelia on permeable supports. Basolateral application to hyperhidrotic cells exhibited a markedly greater increase in Isc than in normal cells. Hyperhidrotic cells also demonstrated differences from the normal in [Ca 2+] i and Isc responses to ATP when pre-treated with thapsigargin. The data demonstrate the presence of apical and basolateral receptors that allow nucleotides to increase [Ca 2+] i and Isc. The results suggest that changes from the normal in transepithelial ion transport contribute to the characteristic excessive fluid production of hyperhidrotic sweat glands.

Neurotensin stimulates Cl− secretion in human colonic mucosa in vitro: Role of adenosine

Background & Aims: Previous studies indicated that the peptide neurotensin (NT) stimulates Cl− secretion in animal small intestinal mucosa in vitro. In this study, we investigated whether NT causes Cl− secretion in human colonic mucosa and examined the mechanism of this response. Methods: Human mucosal preparations mounted in Ussing chambers were exposed to NT. Drugs for pharmacologic characterization of NT-induced responses were applied 30 minutes before NT. Results: Serosal, but not luminal, administration of NT (10−8 to 10−6 mol/L) induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive short-circuit current (Isc) increase that was inhibited by the specific nonpeptide NT receptor antagonists SR 48692 and SR 142948A, the neuronal blocker tetrodotoxin, and the prostaglandin synthesis inhibitor indomethacin. The mast cell stabilizer lodoxamide and the histamine 1 and 2 receptor antagonists pyrilamine and ranitidine, respectively, did not significantly alter NT-induced Isc increase. In contrast, the adenosine receptor 1 and 2 antagonists inhibited this secretory response, whereas the adenosine uptake inhibitors S-(4-nitrobenzyl)-6-thioguanosine and S-(4-nitrobenzyl)-6-thioinosine and the adenosine deaminase inhibitor deoxycoformycin potentiated NT-induced Isc increase. Serosal adenosine induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive Isc increase. Conclusions: NT stimulates chloride secretion in human colon by a pathway(s) involving mucosal nerves, adenosine, and prostaglandins.GASTROENTEROLOGY 2000;119:348-357

UREA MODIFIES THE PERMEABILITY OF THE MAMMALIAN UROTHELIUM

This study investigated the effects of mucosal (urine side) and serosal (blood side) urea on the permeability properties of the in vitro mammalian urinary bladder epithelium. The permeability properties of the rabbit urinary bladder epithelium were studied in modified Ussing chambers using electrophysiological techniques. Addition of two molar urea to the mucosal solution did not cause a significant change in the short circuit current (Isc, a measure of the ion transport capacity of the epithelium), or the transepithelial conductance (Gt, a measure of the ability of ions to diffuse across the epithelium). In contrast, addition of 0.5 M urea to the serosal solution caused an increase in Gt of approximately 35 microS/cm.2 as well as an increase in Isc over a 5 minute period. The site of the conductance increase by short-term serosal urea was at the apical membrane and not at the tight junctions. The urea-induced conductance completely reversed upon removal of urea, was non-selective, and the magnitude was voltage dependent. Long term serosal urea (greater than 30 minutes) resulted in an irreversible increase in transepithelial conductance. Mucosal urea altered the time course but not the magnitude of the serosal urea-induced conductance. The ion permeability of the mammalian urinary bladder is increased by serosal urea. At short times the increase is at the apical membrane, while at long times the increase is at the tight junctions. The presence of mucosal urea slows the loss of urothelial barrier function caused by serosal urea.

Thromboxane A 2-mediated Cl − secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A 2 is a novel endogenous secretagogue of Cl − secretion in the distal colon. Here, we examined if the Cl − secretion caused by platelet-activating factor (PAF; 1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine) is mediated by thromboxane A 2 production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl − secretion in the rat colon. A selective thromboxane A 2 receptor antagonist (sodium( E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[ b, e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[α-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl − current in a concentration-dependent manner. The IC 50 values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E 2-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-( p-chlorobenzyl)-5-(isopropyl)-3- t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl − current. The present study suggests that the PAF-induced Cl − secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A 2.

Effect of alpha1-adrenergic stimulation of Cl- secretion and signal transduction in exocrine glands (Rana esculenta).

In the present work, the effect of stimulation of alpha-adrenergic receptors on Cl- secretion via exocrine frog skin glands was investigated. The alpha-adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the beta-adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl- secretion was measured as the amiloride-insensitive short circuit current (ISC). Addition of noradrenaline induced a biphasic increase in the ISC. The increase in ISC coincided with an increase in the net 36Cl- secretion. The noradrenaline-induced increase in ISC was dose-dependent with an EC50 of 13 +/- 0.3 microM. Epifluorescence microscopic measurements of isolated, fura-2-loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca2+]i) response. Application of noradrenaline induced a biphasic [Ca2+]i response, which was dose-dependent with an EC50 of 11 +/- 6 microM. The Ca2+ plateau unlike the peak-response was sensitive to removal of Ca2+ from the extracellular medium. The noradrenaline-induced increase in the Cl- secretion as well as in [Ca2+]i was sensitive to the alpha1-adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca2+]i indicating that the release was independent of ryanodine-sensitive Ca2+ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5-trisphosphate (IP3) content suggesting that the signal transduction pathway leading to the noradrenaline-induced increase in Ca2+ involved IP3 and a release of Ca2+ from IP3-sensitive stores.

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. Experimental Physiology (2000) 85.3, 255-261.

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.

In situ photon transmission technique for monitoring formation of hydrogels in real-time at various water contents

In situ photon transmission technique was used to monitor the free radical crosslinking copolymerization (FCC) of acrylamide (AAm) and N,N′-methylenebis (acrylamide) (Bis) in real-time. Gelation experiments were performed in various water contents at various wavelengths. It was observed that transmitted photon intensity, Itr, decreased dramatically at a certain gelation time, which is attributed to the increase in scattered light intensity, Isc, during FCC. Increase in Isc was modeled using Rayleigh's equation where gelation time was found to be proportional to the volume of the microgels. The change in Isc is found to be inversely proportional to the thirth power of the wavelength, λ, for high water content samples at early times. However, low water content samples produce smaller and constant power of λ during FCC at all times. The sizes of microgel particles and phase-separated domains were estimated.

ATP stimulates Cl- secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells.

1. Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride-sensitive ISC component. 2. The ATP-induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl- and in the presence of the apical Cl- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 microM) and niflumic acid (100 microM). 3. The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 microM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. 4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated ISC while 'purified' ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT-PCR. 5. Intracellular calcium concentration ([Ca2+]i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 microM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM. 6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na+ absorption and stimulates Cl- secretion via calcium-activated Cl- channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane.

Chloride Secretion in Kidney Distal Epithelial Cells (A6) Evoked by Cadmium

The effect of Cd2+ on chloride secretion was examined in A6 renal epithelia cells by chloride-sensitive fluorescence (SPQ probe) and by the short-circuit-current (Isc) technique. Depleting the cells of Cl− suggests that the Cd2+-activated Isc (ΔIsc(Cd)) is dependent on the presence of Cl− ions. Among the Cl−-channel inhibitors the fenemates, flufenamic acid (FFA) and niflumic acid (NFA), and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) significantly lowered ΔIsc(Cd) compared with control level. In SPQ-loaded A6 cells, Cd2+ evoked an increase in Cl− secretion ([ΔCl−]Cd), which significantly exceeded the basal Cl− transport and was blockable by FFA and NFA. The closely related metals, Zn2+ or Ni2+, were also able to activate Cl− secretion. Preexposure of Zn2+ or Ni2+ completely prevented [ΔCl−]Cd, suggesting that Zn2+ and Ni2+ probably use similar mechanisms. Like Cd2+, thapsigargin (TG), an inhibitor of intracellular Ca2+-ATPase and the Ca2+-ionophore A23187, induced an increase in Isc. Moreover, TG and Cd2+ were able to neutralize the responses of the counterparts as also observed in Isc measurements, which indicates that Cd2+ activates Cl− secretion in a Ca2+-dependent manner. Hence, this study supports the idea that basolateral Cd2+ (possibly also Zn2+ and Ni2+), probably through a Ca2+-sensing receptor, causes calcium mobilization that activates apical fenemate-sensitive chloride channels leading to chloride secretion in A6 cells.

Gamma-Aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.

1. Transintestinal absorption of gamma-aminobutyric acid (GABA) via a pH-dependent mechanism is demonstrated in the model human intestinal epithelial cell line Caco-2. 2. Experiments with BCECF [2',7',-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]-loaded Caco-2 cells demonstrate that GABA transport across the apical membrane is coupled to proton flow into the cell. 3. Short-circuit current (ISC) measurements using Caco-2 cell monolayers under voltage-clamped conditions demonstrate that pH-dependent GABA transport is a rheogenic process even in the absence of extracellular Na+, consistent with H+/GABA symport. 4. A range of GABA analogues were tested for their abilities to: (a) inhibit pH-dependent [3H]GABA uptake across the apical membrane; (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers; (c) increase inward ISC across voltage-clamped Caco-2 cell monolayers. 5. Nipecotic acid, isonipecotic acid, D,L-beta-aminobutyric acid, and 3-amino-1-propanesulphonic acid each caused a marked acidification of intracellular pH and an increase in ISC when superfused at the apical surface of Caco-2 cell monolayers. In contrast L-alpha-amino-n-butyric acid failed to induce proton flow or ISC. The ability of these compounds to induce proton or current flow across the apical surface of this intestinal epithelium was closely related to the relative inhibitory effects on [3H]GABA uptake. 6. These observations demonstrate H+/GABA symport and suggest that this transport mechanism may be accessible as a route for oral absorption of therapeutically-useful GABA analogues.

Ontogenic aspects of D1 receptor coupling to G proteins and regulation of rat jejunal Na+, K+ ATPase activity and electrolyte transport.

1. The present study examined the effect of dopamine on rat jejunal electrolyte transport (rheogenic transport and Na+, K(+)-ATPase activity) in adult (60-day old) and young (20-day old) animals. 2. In young rats, dopamine, in the presence of phentolamine, produced an increase in jejunal Isc, this being completely abolished by SKF 83566, and not changed by S-sulpiride. SKF 38393, but not quinerolane, also increased Isc; this effect was abolished by SKF 83566 and ouabain, but not by furosemide. In adult rats, dopamine in the presence of phentolamine (0.2 microM) decreased Isc. 3. Na+, K(+)-ATPase activity in isolated jejunal epithelial cells from adult rats was 2.4 fold that in young rats. In the presence of phentolamine, both dopamine and SKF 38393, but not quinerolane, significantly decreased jejunal Na+, K(+)-ATPase activity in young animals but not in adult animals. 4. Binding [3H]-Sch 23390 to membranes of jejunal mucosa revealed the presence of a single class of receptors in both young and adult rats, with similar KD and Bmax values. 5. GTP gamma S and cholera toxin inhibited jejunal Na+, K(+)-ATPase activity in young, but not in adult rats. Co-incubation of pertussis toxin with dopamine was found to potentiate the inhibitory effects of dopamine upon the enzyme in both young and adult rats. 6. Regulation of Na+, K(+)-ATPase activity by cholera toxin-sensitive G proteins is absent in adult animals, and such difference may explain the failure of dopamine to inhibit intestinal Na+, K(+)-ATPase activity in adult rats.

Stimulation of Cl(-) secretion by L-alanine oligopeptides in the mammalian large intestine.

A variety of oligopeptides are probably released within the intestinal tissue under inflammatory conditions or during peptide absorption. To examine whether some of these peptides can affect intestinal transport functions, we determined the effects of L-alanine oligopeptide on short-circuit current (I(sc)) and transmucosal conductance (G(t)) in submucosa-mucosa preparations from the mouse cecum and guinea pig distal colon in vitro in Ussing chambers. L-Alanyl-L-alanine (Ala-Ala, 10 mM) added to the serosal side increased I(sc) and G(t), giving a peak followed by a sustained phase (the peak increase in I(sc) was 45 +/-6 microA/cm(2) and the increase in G(t) was 0.55+/-0.11 mS/cm(2)). The tripeptide, L-alanyl-L-alanyl-L-alanine (Ala-Ala-Ala, 10 mM), added to the serosal side also induced increases in I(sc) and G(t) by a similar degree. On the other hand, luminal Ala-Ala, and serosal L-alanine and L-alanine (10 mM) caused significantly smaller increases in I(sc) and G(t) ( approximately 15 microA/cm(2) and approximately 0. 15 mS/cm(2), respectively). The Ala-Ala induced increase in I(sc) was partially inhibited by serosal bumetanide (0.1 mM) and mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid (0.1 mM), and largely suppressed by removing Cl(-) from the bathing solution. The increase in I(sc) was largely suppressed by serosal low Ca(2+) and tetrodotoxin, but was not affected by indomethacin. In the guinea pig distal colon, serosal Ala-Ala (10 mM) evoked a transient increase in I(sc) by 23+/-7 microA/cm(2) and an increase in G(t) by 1.2+/-0.3 mS/cm(2). These results suggest that Ala-Ala, and probably also Ala-Ala-Ala, added to the serosal side stimulated electrogenic Cl(-) secretion mainly through the activation of submucosal secretomotor neurons in the mammalian large intestine.

Effects of Higenamine on Regulation of Ion Transport in Guinea Pig Distal Colon

Effects of higenamine on Na+, K+ and Cl- transport were studied on stripped guinea pig distal colonic mucosa in vitro using Ussing chambers. Addition of 10(-5) M higenamine induced a biphasic change in short circuit current (Isc): a transient increase followed by a long-lasting decrease that was accompanied by an increase in transepithelial conductance (Gt). The initial phase with an increase in Isc was partially inhibited by serosal bumetanide and abolished by mucosal diphenylamine-2-carboxylate, a chloride channel blocker, indicating transient induction of Cl- secretion. The second phase with a decrease in Isc was composed of two effects: the inhibition of the amiloride-sensitive electrogenic Na+ absorption and the stimulation of the bumetanide-sensitive K+ secretion. However, the initial transient increase was not observed at the lower concentration of higenamine (10(-8)-10(-6) M). All the changes in Isc and Gt induced by higenamine were suppressed by the non-selective beta-adrenergic receptor antagonist propranolol and by the beta2-adrenergic receptor antagonist ICI-118,551, but not by the beta1-adrenergic-receptor-selective antagonist atenolol or by the alpha-antagonists phentolamine, prazosin and yohimbine. These results suggest that higenamine inhibits electrogenic Na+ absorption and stimulates electrogenic K+ and Cl- secretion through beta2-adrenergic receptors in guinea pig distal colon.

Effects of dietary substitution with raw and heat-treated cowpea (Vigna unguiculata) on intestinal transport and pancreatic enzymes in the pig.

Tropical grain legumes represent potentially important feed for farm animals. However, diarrhoea and poor growth performance have been reported, due to the various anti-nutritional factors they contain. This study addressed in particular whether dietary cowpea impaired the growth of pigs, whether the small intestinal Na+/D-glucose coabsorptive transport capacity was decreased, whether the Cl- secretory capacity was increased, and, finally, whether these parameters were affected by heat treatment of cowpea. Pigs, 4 weeks old, were fed for 3 weeks with one of three diets: (i) standard soy, (ii) 75% of soy substituted with raw cowpea, or (iii) 75% of soy substituted with heat-treated cowpea. The absorptive and secretory capacities of the jejunum and ileum were measured with the Ussing chamber technique. Weight gain, feed intake, pancreatic protein and enzyme concentrations and levels of the blood hormones glucagon and cholecystokinin were also measured. The Na+ transport capacity was measured as the increase in short-circuit current (Isc) when D-glucose was added to the luminal side in the Ussing chambers. Isc was significantly higher in the jejunum from raw cowpea-fed pigs than in the jejunum from standard soy-fed pigs, with no difference between the two cowpea-fed groups. The phosphodiesterase inhibitor theophylline was subsequently added bilaterally, and the increase in Isc indicated the cAMP-depedent Cl- secretory capacity. In the jejunum this was significantly higher in raw and heat-treated cowpea-fed pigs than in standard soy-fed pigs. In contrast, there were no differences in the ileal transport capacities. There were no differences in the pancreatic protein and trypsin concentrations or the blood hormones, but the raw cowpea-fed pigs had significantly lower pancreatic amylase than standard soy-fed pigs. Weight gain and feed intake were lowest in the cowpea-fed groups, with no significant difference between the two groups. In conclusion, the hypothesis of impaired small intestinal absorption of D-glucose and Na+ as causing malabsorption, and therefore impaired growth, during cowpea substitution in the feed may be firmly rejected. The increased Cl- secretory capacity, although moderate, may contribute to the higher incidence of post-weaning diarrhoea in cowpea-fed pigs, as observed in other studies. Additionally, the decreased food intake, feed conversion and weight gain were unaffected by heat treatment, further suggesting involvement of heat-stable anti-nutritional factors.

Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells.

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.

The Simultaneous Measurement of Epithelial Ion Transport and Intracellular Free Ca 2+ in Cultured Equine Sweat Gland Secretory Epithelium

We explored the relationship between nucleotide-evoked changes in intracellular free calcium ([Ca2+]i) and anion secretion by measuring [Ca2+]i and I(SC) simultaneously in Fura-2-loaded, cultured equine sweat gland epithelia. Apical ATP, UTP or UDP elicited sustained increases in [Ca2+]i that were initiated by the mobilization of cytoplasmic Ca2+ but maintained by Ca2+ influx. However, although these nucleotides also increased I(SC), this response was transient whereas the [Ca2+]i signals were sustained. Experiments in which external Ca2+ was removed/replaced showed that Ca2+ entering nucleotide-stimulated cells elicited very little change in ISC. Cross desensitization experiments showed that UTP-stimulated epithelia became insensitive to ATP but that UTP could increase both [Ca2+]i and ISC in ATP-stimulated cells by activating 'pyrimidinoceptors' essentially insensitive to ATP. Thapsigargin evoked a sustained rise in [Ca2+]i that was accompanied by a maintained increase in ISC. However, this increase in ISC was dependent upon external Ca2+ and so the responses to nucleotides and thapsigargin have different properties. ATP increased ISC in thapsigargin-treated cells without causing any rise in [Ca2+]i while ionomycin increased both parameters. The data therefore show that apical P2Y receptors allow nucleotides to increase ISC via two mechanisms, one of which appears to be [Ca2+]i-independent control of anion channels.

Electrical properties of the intestinal mucosa of the chicken and the effects of luminal glucose

Transmural potential difference (PD), short-circuit current (Isc), and electrical resistance (R) were measured in the isolated mucosa of the duodenum, jejunum, ileum, proximal cecum, and rectum in order to characterize the electrical properties of the chicken small and large intestine. The chicken intestine was classified into three categories, regarding its electrical characteristics: 1) the duodenum, with four to five times higher R than the other segments and the lowest PD; 2) the group formed by the jejunum, the ileum, and the proximal cecum, with high PD and low R; 3) the rectum, with low PD and low R. In all segments, the addition of D-glucose into the luminal side stimulates Isc, and this effect can be reversed by phloridzin, indicating that the glucose-induced Isc increase is due to Na+-D-glucose co-transport. The effect of glucose is maximal in the rectum, with a fivefold Isc increase, suggesting that this segment may have an important role in the absorption of Na+ as well as of nutrients co-transported with Na+.

Forskolin increases apical sodium conductance in cultured toad kidney cells (A6) by stimulating membrane insertion.

The role of membrane traffic in the stimulation of apical Na+ permeability caused by increases in cytoplasmic cyclic AMP was assessed by measuring the effects of forskolin on transepithelial capacitance (CT), transepithelial conductance (GT), and short-circuit current (Isc) in A6 cultured toad kidney cells. Apical water permeability was probed by recording cell volume changes after reducing the osmolality of the apical bath. We found that forskolin does not increase the osmotic water permeability of the apical membrane of A6 cells, and thus does not stimulate the insertion of water channels. Comparison of the effects of forskolin and insulin on Na+ transport demonstrated that both agents produce reversible increases in CT, GT and Isc. GT and CT increased proportionally during the rising phase of the insulin response. However, a non-linear relationship between both parameters was recorded when forskolin was given in NaCl Ringer's solution. The relationship between CT and GT became linear after the effects of forskolin on Cl- conductances were eliminated by substituting Cl- by an impermeant anion. In contrast, in Cl--containing Na+-free solutions, the non-linearity became more pronounced. Successive additions of insulin and forskolin caused additive increases in CT. Because increases in CT and Na+ transport occurred in the absence of stimulation of water permeability and increases of CT and GT were directly proportional when Na+ was the major permeating ion across the apical membrane, we suggest that the increase in apical Na+ permeability in the presence of either forskolin or insulin is due to the insertion of channels residing in intracellular pools. In contrast, the increased Cl- permeability caused by forskolin may be related to the activation of channels already present in the membrane.