Localization of sodium absorption and chloride secretion in an intestinal epithelium

Summary Hen coprodeum absorbs sodium electrogenically and, when stimulated by theophylline, secretes chloride. In this study the vibrating microprobe technique was used to localize the transport of these ions to intestinal villi/folds and crypts. With the isolated, stretched epithelium, controlled by light microscopy and scanning electron microscopy, in open circuit, currents were inward, 40±7 A/cm², 50 m vertically above villi, and outward, 36±7 A/cm² above crypts. The currents decayed exponentially to near zero at 300 m with the same length constant. A physical model simulating the observed loci of current sources and sinks predicts potential profiles consistent with our data. Extrapolation of the currents gives a surface potential of 45 V, negative on villi and positive above crypts. Short circuiting increased villus current to 86±27 A/cm² at 50 m, and amiloride treatment reduced it to –8 A/cm²; in both cases crypt currents were abolished. The inward currents are compatible with sodium absorption. Induction of chloride secretion after amiloride treatment, resulted in current circuits similar to those induced by sodium absorption, with villus currents of 23±7 A/cm². This is in accord with chloride secretion at the villi. Quantitative estimates of crypt number (860/cm²) and opening diameter (15 m), in conjunction with isotopic measurements of active and electrical potential-driven ion fluxes demonstrate, however, that only 4% of the potential-driven co-ion transport occurs through the crypts. This indicates that nearly all chloride secretion comes from the sodium-absorbing villar area. Were the chloride secretion to occur solely from the crypts, the current should have been in the opposite direction and 10,000-fold larger.     

Membrane phospholipid composition affects function of potassium channels from rabbit colon epithelium

We tested the effects of membrane phospholipids on the functionof high-conductance,Ca²⁺-activatedK⁺ channels from the basolateralcell membrane of rabbit distal colon epithelium by reconstituting thesechannels into planar bilayers consisting of different 1:1 mixtures ofphosphatidylethanolamine (PE), phosphatidylcholine (PC),phosphatidylserine (PS), and phosphatidylinositol (PI). At low ambientK⁺ concentrations single-channelconductance is higher in PE/PS and PE/PI bilayers than in PE/PCbilayers. At high K⁺concentrations this difference in channel conductance is abolished. Introducing the negatively charged SDS into PE/PC bilayersincreases channel conductance, whereas the positively chargeddodecyltrimethylammonium has the opposite effect. All these findingsare consistent with modulation of channel current by the charge of thelipid membrane surrounding the channel. But theK⁺ that permeates the channelsenses only a small fraction of the full membrane surface potential ofthe charged phospholipid bilayers, equivalent to separation of theconduction pathway from the charged phospholipid head groups by 20 Å. This distance appears to insulate the channel entrancefrom the bilayer surface potential, suggesting large dimensions of thechannel-forming protein. In addition, in PE/PC and PE/PI bilayers, butnot in PE/PS bilayers, the open-state probability of the channeldecreases with time ("channel rundown"), indicating thatphospholipid properties other than surface charge are required tomaintain channel fluctuations.

     

Intracellular activities of chloride, potassium and sodium ions in rabbit corneal epithelium

The mechanism of ion transport in the epithelium of rabbit cornea was studied by determining the intracellular ion activity of Cl-, Na+ and K+ under various conditions. Ionic activities were measured by means of microelectrodes containing liquid ion-exchangers selective for Cl-, Na+ or K+. The Cl- activity in basal cells of the epithelium in Na+ containing bathing solutions amounts to 28 +/- 2 mM (n = 11). This value is 1.9-times greater than expected on the basis of passive distribution across the tear side membrane. This finding suggests the existence of a Cl- accumulating process. Replacement of Na+ in the aqueous bathing solution by choline or tetraethylammonium results in a reversible decrease in Cl- activity to 22 +/- 1 mM (n = 11, P less than 0.025). The ratio of observed and predicted Cl- activity decreased significantly from 1.9 to 1.4 (P less than 0.05). The decrease in Cl- activity due to Na+ replacement was rather slow. In contrast, after readmittance of Na+ to the aqueous bathing solution, Cl- activity rose to a stable level within 30 min. These results indicate involvement of Na+ in Cl- accumulation into the basal cells of the epithelium. The K+ and Na+ activities of the basal cells of rabbit corneal epithelium in control bathing solutions were 75 +/- 4 mM (n = 13) and 24 +/- 3 mM (n = 12), respectively. The results can be summarized in the following model for Cl- transport across corneal epithelium. Cl- is accumulated in the basal cells across the aqueous side membrane, energized by a favourable Na+ gradient. Cl- will subsequently leak out across the tear side membranes. Na+ is extruded again across the aqueous side membrane of the epithelium by the (Na+ + K+)-ATPase.     

Active potassium transport by rabbit descending colon epithelium

Summary Previous studies of rabbit descending colon have disagreed concerning potassium transport across this epithelium. Some authors reported active K⁺ secretion underin vitro short-circuited conditions, while others suggested that K⁺ transport occurs by passive diffusion through a highly potassium-selective paracellular route. For this reason, we re-examined potassium fluxes across the colon in the presence of specific and general metabolic inhibitors. In addition, electrochemical driving forces for potassium across the apical and basolateral membranes were measured using conventional and ion-sensitive microelectrodes. Under normal conditions a significant net K⁺ secretion was observed (J _net ^K =–0.39±0.081 eq/cm²hr) with⁴²K fluxes, usually reaching steady-state within approximately 50 min following isotope addition. In colons treated with serosal addition of 10^–4 m ouabain,J _sm ^K was lowered by nearly 70% andJ _ms ^K was elevated by approximately 50%. Thus a small but significant net absorption was present (J _net ^K =0.12±0.027 eq/cm²hr). Under control conditions, the net cellular electrochemical driving force for K⁺ was 17 mV, favoring K⁺ exit from the cell. Cell potential measurements indicated that potassium remained above equilibrium after ouabain, assuming that passive membrane permeabilities are not altered by this drug. Net K⁺ fluxes were abolished by low temperature.The results indicate that potassium transport by the colon may occur via transcellular mechanisms and is not solely restricted to a paracellular pathway. These findings are consistent with our previous electrical results which indicated a nonselective paracellular pathway. Thus potassium transport across the colon can be modeled as a paracellular shunt pathway in parallel with pump-leak systems on the apical and basolateral membranes.     

The sodium and chloride dependence of chloride secretion by the opercular epithelium

The effects of ion substitutions on the Cl- secretion rate and tissue conductance of isolated short-circuited opercular epithelia from sea-water-adapted Fundulus heteroclitus were investigated. Serosal Na+ substitution had the same effect on the Cl- secretion rate that serosal Cl- substitution had on the active component of the Cl- efflux. This similarity indicated a 1:1 Na-Cl requirement for active Cl- secretion across this epithelium, which supports the proposal of a coupled NaCl uptake mechanism at the serosal membrane of Cl- secretory epithelia. Mucosal Na+ and Cl- substitutions appeared to inhibit completely the active Cl- secretory flux. The reductions in the tissue conductance with mucosal ion substitutions suggested that this effect can be attributed to a blocking of the apical membrane Cl- conductance. These mucosal ion effects suggested a possible direct regulatory influence of the external salinity on the Cl- secretion rate and tissue conductance, which provide alternative explanations for observations with the teleost gill epithelium.     

Vanadium ion stimulation of chloride secretion by rabbit colonic epithelium

Ionized forms of vanadium are known to exert diverse biological activities. Of particular interest in the inhibitory action of the vanadium ion on (Na+ + K+)-ATPase. This report describes another action of the vanadium ion on the rabbit colonic epithelium. Micromolar quantities of vanadate, applied to the serosal side of the isolated rabbit colonic epithelium, result in a stimulation of electrogenic chloride secretion by this epithelium. Sodium transport is unaffected by the vanadium ion in the concentrations used in this study. It is proposed that the vanadyl ion activates adenylate cyclase and thereby initiates subsequent secretory events.     

Basolateral K-Cl cotransporter regulates colonic potassium absorption in potassium depletion

Active potassium absorption in the rat distal colon is electroneutral, Na(+)-independent, partially chloride-dependent, and energized by an apical membrane H,K-ATPase. Both dietary sodium and dietary potassium depletion substantially increase active potassium absorption. We have recently reported that sodium depletion up-regulates H,K-ATPase alpha-subunit mRNA and protein expression, whereas potassium depletion up-regulates H,K-ATPase beta-subunit mRNA and protein expression. Because overall potassium absorption is non-conductive, K-Cl cotransport (KCC) at the basolateral membrane may also be involved in potassium absorption. Although KCC1 has not been cloned from the colon, we established, in Northern blot analysis with mRNA from the rat distal colon using rabbit kidney KCC1 cDNA as a probe, the presence of an expected size mRNA in the rat colon. This KCC1 mRNA is substantially increased by potassium depletion but only minimally by sodium depletion. KCC1-specific antibody identified a 155-kDa protein in rat colonic basolateral membrane. Potassium depletion but not sodium depletion resulted in an increase in KCC1 protein expression in basolateral membrane. The increase of colonic KCC1 mRNA abundance and KCC1 protein expression in potassium depletion of the rat colonic basolateral membrane suggests that K-Cl cotransporter: 1) is involved in transepithelial potassium absorption and 2) regulates the increase in potassium absorption induced by dietary potassium depletion. We conclude that active potassium absorption in the rat distal colon involves the coordinated regulation of both apical membrane H,K-ATPase and basolateral membrane KCC1 protein.     

Basolateral Cl- channels in the larval bullfrog skin epithelium

The addition of 150 U/ml nystatin to the mucosal surface of isolated skin from larval bullfrogs increases apical membrane permeability and allows a voltage clamp to be applied to the basolateral membrane. With identical Ringer's solutions bathing either side of the tissue the short-circuit current (I(SC)) averaged 7.60+/-0.78 micro A/cm2, and this current could be increased or decreased by imposing a Cl- concentration gradient. Fluctuation analysis of the I(SC) gave power spectra that could be fit with low- and high-frequency Lorentzian functions having corner frequencies of 1.48+/-0.06 Hz and 48.5+/-11.4 Hz, respectively. The Lorentzian plateau was minimal at the lowest I(SC) and increased as the I(SC) became greater in the positive or negative direction. Current-voltage plots with identical Ringer's on either side of the tissue showed a pattern of outward rectification. Cell attached patches of cells isolated from the skin with collagenase-trypsin treatment showed spontaneous channel activity with a conductance of 20.9 pS at a pipette potential, -Vp=20 mV. Current-voltage plots of single channels showed a similar pattern of rectification to that of the intact skin, and partial replacement of Cl- by gluconate in the pipette solution shifted the reversal potential from zero to about 40 mV, which is close to the expected shift of the reversal potential of the chloride current through a Cl- selective ion channel. These results suggest that the basolateral Cl- conductance of the larval skin is mediated by a channel with properties that resemble a volume-sensing outward-rectifier anion channel that has been described in a variety of cell types     

Basolateral membrane potassium conductance is independent of sodium pump activity and membrane voltage in canine tracheal epithelium

Summary When secretagogues stimulate Cl secretion in canine tracheal epithelium, apical membrane Cl conductance (G _a ^Cl ) increases, and then basolateral membrane K conductance (G _b ^K ) increases. Conversely, inhibition ofG _a ^Cl results in a secondary decrease inG _b ^K . The coordination of the two membrane conductances and regulation ofG _b ^K is critical for maintaining constant intracellular ion concentrations and transepithelial Cl secretion. The purpose of this study was to test two hypotheses about the regulation ofG _b ^K . First, we asked whetherG _b ^K is directly linked to the activity of the Na,K-ATPase. We found that pump activity could be dissociated from K conductance. Inhibition of the Na pump with ouabain, in nonsecreting tissues led to an increase inG _b . Elevation of the bathing solution K concentration produced a similar effect. Addition of ouabain to secreting tissues did not appear to alterG _b . These results indicate thatG _b ^K does not directly parallel Na pump activity. Second, we asked whether changes inG _b ^K are voltage dependent. We prevented secretagogue-induced depolarization of the electrical potential difference across the basolateral membrane _b by clamping _b at its resting value during stimulation of Cl secretion with epinephrine. Despite maintaining _b constant, the typical changes in transepithelial resistance and the ratio of membrane resistances persisted. This observation indicates that depolarization is not required for the secretagogue-induced increase inG _b ^K . In addition we examined the effect of depolarizing and hyperpolarizing _b by passing transepithelial current in secreting and nonsecreting epithelia. Despite depolarizing and hyperpolarizing _b within the physiologic range, we observed no significant changes in transepithelial resistance or the ratio of membrane resistance that would suggest a change inG _b ^K . This observation indicates that changes in _b are not sufficient to alterG _b ^K . Thus,G _b ^K appears to be regulated by factors other than membrane voltage, or direct coupling to the Na pump.     

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.     

Steady-state sodium absorption and chloride secretion of colon and coprodeum,and plasma levels of osmoregulatory hormones in hens in relation to sodium intake

Summary The plasma levels of four osmoregulatory hormones and their target ion-transport systems in the lower intestines of the domestic fowl were determined in order to elucidate their interrelationship and their setpoints in relation to NaCl intake. White Plymouth Rock hens were adapted to six intake levels of NaCl (0.20±0.02–24.7±1.9 mmoles Na⁺·kg bw^–1·day^–1) for 6 weeks. The Na⁺ absorption and the Cl^– secretion of colon and coprodeum were characterized in vitro by the effects of hexoses, amino acids, amiloride, and theophylline on the short-circuit current (SCC) and electrical potential difference (PD). The NaCl-conserving system of the adult chicken is set at low intake levels of NaCl as the 80% range (quantitized by non-linear, logistic regression analyses) of the change in the plasma [ALDO], the amiloride-inhibitable Na⁺ absorption of coprodeum and colon ( SCC), occurred from 0.18 to 2.3, from 0.9 to 4.3, and from 1.2 to 7.3 mmoles Na⁺·kg bw^–1·day^–1, respectively. These results demonstrate that the amiloride-inhibitable Na⁺ absorption of coprodcum is more closely linked to plasma [ALDO] than that of colon. The aminoacid-Na⁺ coabsorption of colon increased over exactly the same range of Na⁺ intake as the colonic amiloride-inhibitable Na⁺ absorption decreased, whereas the hexose-Na⁺ coabsorption increased at higher levels of Na⁺ intake, from 2 to 11 mmoles Na⁺·kg bw^–1·day^–1. Both these Na⁺ absorption types had reached their maximums at 24.7 mmoles Na⁺·kg bw^–1·day^–1, whereas the plasma [AVT] and plasma [PRL], although significantly increased, apparently had not; their 80% range of change occurred from 9.9 to 99 mmoles Na⁺·kg bw^–1·day^–1, and the main changes in plasma osmolity were predicted to occur from 5.4 to 107 mmoles Na⁺·kg bw^–1·day^–1. These results suggest that these colonic and hormonal variables conserve osmotically-free water and operate at high NaCl intake. The theophylline-induced colonic Cl^– secretion did not change with NaCl intake, whereas the stimulation of SCC in coprodeum decreased with increasing NaCl intake: The main change occurred between 0 and 3.2 mmoles Na⁺·kg bw^–1·day^–1. Thus, all ion-transport capacity disappears in coprodeum with increased dietary NaCl intake, whereas colon maintains its ion-transport capacity (although the nature of the Na⁺ transport changes). It is suggested that hormones defending the extracellular volume and composition are regulated close to zero input and output of both NaCl and water, regardless of whether they are NaCl conserving or free-water conserving. Therefore, changes in their stable plasma concentrations occur at the extremes of tolerable range of NaCl intake.Abbreviation AA aminoacids - ALDO aldosterone - AMI amiloride - AVT arginine vasotocin - bw body weight - CS corticosterone - HEX hexoses - INDO indomethacin - PD potential difference - PRL prolactin - R resistance - SCC short-circuit current - SD standard deviation - SEM standard error of mean - THEO theophylline     

Mitochondrial potassium and chloride channels

Channels selective for potassium or chloride ions are present in inner mitochondrial membranes. They probably play an important role in mitochondrial events such as the formation of delta pH and regulation of mitochondrial volume changes. Mitochondrial potassium and chloride channels could also be the targets for pharmacologically active compounds such as potassium channel openers and antidiabetic sulfonylureas. This review describes the properties, pharmacology, and current observations concerning the functional role of mitochondrial potassium and chloride channels.     

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.     

Multiple potassium and chloride channels in the human colon carcinoma cell line SW1116

SW1116 cells have a profound capacity for secreting mucin molecules bearing the Lewisa epitope. Mucin molecules with the same epitope have been found to be elevated in the serum of patients with cystic fibrosis, a disease with defective ion channels. We therefore decided to study ion channels in this cell line. In the present work, we report the presence of two K(+)-channels and two Cl(-)-channels in the apical membrane of SW1116 cells. One of the K(+)-channels has a large conductance (approximately 278 pS), anomalous rectifying properties, and is inactivated rapidly. The second type exhibited a linear I/V curve (19 pS), was voltage insensitive and inactivation was not observed. In cell-attached patches, spontaneous openings of chloride channels were seen with higher frequency than previously reported in other colon carcinoma cell lines or airway epithelial cells. Inside-out experiments allowed identification of two different Cl(-)-channels (Cl(-)-1 and Cl(-)-2). Both exhibited rectification, but in opposite directions, and both were insensitive to NIPAB.     

The presence of voltage-gated sodium, potassium and chloride channels in rat cultured astrocytes

Patch-clamp recording from the plasmalemma of rat cultured astrocytes reveals the presence of both voltage-dependent sodium and voltage-dependent potassium conductances. These conductances are similar but not identical to the corresponding conductances in the axolemma. Whereas the h infinity relation of the sodium channels has the same voltage dependence as in the nodal axolemma, the peak current-voltage relation is shifted by about 30 mV along the voltage axis in the depolarizing direction. It is speculated that the glial cells synthesize sodium and potassium channels for later insertion into the axolemma of neighbouring axons. The astrocytes also express a plasmalemmal voltage-dependent anion conductance that is turned on at about -40 mV (that is, near the resting potential of the cultured astrocytes). The channels involved are large enough to be just permeable to glutamate but not to ascorbate. It is suggested that the conductance of this channel for chloride plays a physiological role in the spatial buffering of potassium by glial cells.