K+ transport and capacitance of the basolateral membrane of the larval frog skin

Skin from larval bullfrogs was mounted in an Ussing-type chamberin which the apical surface was bathed with a Ringer solution containing 115 mM K⁺ and thebasolateral surface was bathed with a Ringer solution containing 115 mMNa⁺. Ion transport was measured asthe short-circuit current(I_sc) with alow-noise voltage clamp, and skin resistance(R_m) wasmeasured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine wavesto the command stage of the voltage clamp. From the ratio of theFourier-transformed voltage and current signals, it was possible tocalculate the resistance and capacitance of the apical and basolateralmembranes of the epithelium(R_a andR_b,C_a and C_b,respectively). With as the anion,R_m decreasedrapidly within 5 min following the addition of 150 U/ml nystatin to theapical solution, whereasI_sc increasedfrom 0.66 to 52.03 µA/cm² over a60-min period. These results indicate that nystatin becomes rapidlyincorporated into the apical membrane and that the increase inbasolateral K⁺ permeabilityrequires a more prolonged time course. Intermediate levels ofI_sc were obtainedby adding 50, 100, and 150 U/ml nystatin to the apical solution. Thisproduced a progressive decrease in R_a andR_b whileC_a andC_b remainedconstant. With Cl as theanion, I_sc valuesincreased from 2.03 to 89.57 µA/cm² following treatment with150 U/ml nystatin, whereas with gluconate as the anionI_sc was onlyincreased from 0.63 to 11.64 µA/cm². This suggests that theincrease in basolateral K⁺permeability produced by nystatin treatment, in the presence of morepermeable anions, is due to swelling of the epithelial cells of thetissue rather than the gradient for apicalK⁺ entry. Finally,C_b was notdifferent among skins exposed toCl,, or gluconate, despite the largedifferences inI_sc, nor didinhibition of I_scby treatment with hyperosmotic dextrose cause significant changes inC_b. These resultssupport the hypothesis that increases in cell volume activateK⁺ channels that are alreadypresent in the basolateral membrane of epithelial cells.

     

Cryptdin-3 induces novel apical conductance(s) in Cl- secretory, including cystic fibrosis, epithelia

Opening ofanion-conductive pathways in apical membranes of secretory cells liningmucosal surfaces is a critical step in salt and water secretion and,thus, hydration of sites including airway and intestine. In intestine,Paneth cells are positioned at the base of the secretory gland (crypt)and release defensin peptide, in mice termed cryptdins, into the cryptlumen. Because at least some defensins have been shown to formanion-conductive channels in phospholipid bilayers, we tested whetherthese endogenous antimicrobial peptides could act as soluble inducersof channel-like activity when applied to apical membranes. To directlyevaluate the possibility of cryptdin-3-mediated apical anionconductance (G_ap), we have utilized amphotericinB to selectively permeabilize basolateral membranes of electricallytight monolayers of polarized human intestinal secretory epithelia (T84cells), thus isolating the apical membrane for study. Cryptdin-3induces G_ap that is voltage independent(G_ap = 1.90 ± 0.60 mS/cm²) and exhibits ion selectivity contrasting to thatelicited by forskolin or thapsigargin (for cryptdin-3,Cl = gluconate; for forskolin and thapsigargin,Cl gluconate). We cannot exclude the possibility thatthe macroscopic current induced by cryptdin could be the sum of cationand Cl currents. Cryptdin-3 induces a current inbasolaterally permeabilized epithelial monolayers derived from airwaycells harboring the F508 mutation of cystic fibrosis (CF;G_ap = 0.80 ± 0.06 mS/cm²), demonstrating that cryptdin-3 restores anionsecretion in CF cells; this occurs independently of the CFtransmembrane conductance regulator channel. These results support theidea that cryptdin-3 may associate with apical membranes ofCl-secreting epithelia and self-assemble into conductingchannels capable of mediating a physiological response.

     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.

     

Dexamethasone treatment causes resistance to insulin-stimulated cellular potassium uptake in the rat

Patients treated with glucocorticoids have elevated skeletal muscle ouabain binding sites. The major Na⁺-K⁺-ATPase (NKA) isoform proteins found in muscle, ₂ and ₁, are increased by 50% in rats treated for 14 days with the synthetic glucocorticoid dexamethasone (DEX). This study addressed whether the DEX-induced increase in the muscle NKA pool leads to increased insulin-stimulated cellular K⁺ uptake that could precipitate hypokalemia. Rats were treated with DEX or vehicle via osmotic minipumps at one of two doses: 0.02 mg·kg^–1·day^–1 for 14 days (low DEX; n = 5 pairs) or 0.1 mg·kg^–1·day^–1 for 7 days (high DEX; n = 6 pairs). Insulin was infused at a rate of 5 mU·kg^–1·min^–1 over 2.5 h in conscious rats. Insulin-stimulated cellular K⁺ and glucose uptake rates were assessed in vivo by measuring the exogenous K⁺ infusion () and glucose infusion (G_inf) rates needed to maintain constant plasma K⁺ and glucose concentrations during insulin infusion. DEX at both doses decreased insulin-stimulated glucose uptake as previously reported. G_inf (in mmol·kg^–1·h^–1) was 10.2 ± 0.6 in vehicle-treated rats, 5.8 ± 0.8 in low-DEX-treated rats, and 5.2 ± 0.6 in high-DEX-treated rats. High DEX treatment also reduced insulin-stimulated K⁺ uptake. (in mmol·kg^–1·h^–1) was 0.53 ± 0.08 in vehicle-treated rats, 0.49 ± 0.14 in low-DEX-treated rats, and 0.27 ± 0.08 in high-DEX-treated rats. DEX treatment did not alter urinary K⁺ excretion. NKA ₂-isoform levels in the low-DEX-treated group, measured by immunoblotting, were unchanged, but they increased by 38 ± 15% (soleus) and by 67 ± 3% (gastrocnemius) in the high-DEX treatment group. The NKA ₁-isoform level was unchanged. These results provide novel evidence for the insulin resistance of K⁺ clearance during chronic DEX treatment. Insulin-stimulated cellular K⁺ uptake was significantly depressed despite increased muscle sodium pump pool size. skeletal muscle; sodium pump; Na⁺-K⁺-ATPase     

Nerve growth factor regulates HCO-3 absorption in thick ascending limb: modifying effects of vasopressin

Growth factorsstimulateNa⁺/H⁺exchange activity in many cell types but their effects on acidsecretion via this mechanism in renal tubules are poorly understood. Weexamined the regulation of HCO₃absorption by nerve growth factor (NGF) in the rat medullary thickascending limb (MTAL), which absorbs HCO₃via apical membraneNa⁺/H⁺exchange. MTAL were perfused in vitro with 25 mMHCO₃ solutions (pH 7.4; 290 mosmol/kgH₂O). Addition of 0.7 nMNGF to the bath decreased HCO₃absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol · min¹ · mm¹(P < 0.001). In contrast, with10¹⁰ M arginine vasopressin(AVP) in the bath, addition of NGF to the bath increasedHCO₃ absorption from 8.0 ± 1.6 to12.5 ± 1.3 pmol · min¹ · mm¹(P < 0.01). Both effects of NGF wereblocked by genistein, consistent with the involvement of tyrosinekinase pathways. However, the AVP-dependent stimulation requiredactivation of protein kinase C (PKC), whereas the inhibition was PKCindependent, indicating that the NGF-induced signaling pathways leadingto inhibition and stimulation of HCO₃absorption are distinct. Hypertonicity blocked the inhibition but notthe AVP-dependent stimulation, suggesting that hypertonicity and NGFmay inhibit HCO₃ absorption via acommon mechanism. These data demonstrate that NGF inhibitsHCO₃ absorption in the MTAL underbasal conditions but stimulates HCO₃ absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is acritical determinant of the response of the MTAL to growth factorstimulation and suggest that NGF can either inhibit or stimulateapical Na⁺/H⁺ exchange activitydepending on its interactions with other regulatory factors. Locallyproduced growth factors such as NGF may play a role in regulating renaltubule HCO₃ absorption.

     

Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter

We have clonedand functionally characterized the human Na⁺-dependenthigh-affinity dicarboxylate transporter (hNaDC3) from placenta. ThehNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the clonedtransporter mediates the transport of succinate in the presence ofNa⁺ [concentration of substrate necessary for half-maximaltransport (K_t) for succinate = 20 ± 1 µM]. Dimethylsuccinate also interacts with hNaDC3. TheNa⁺-to-succinate stoichiometry is 3:1 and concentration ofNa⁺ necessary for half-maximal transport(K^Na+_0.5) is 49 ± 1 mM as determined by uptake studies withradiolabeled succinate. When expressed in Xenopuslaevis oocytes, hNaDC3 induces Na⁺-dependent inwardcurrents in the presence of succinate and dimethylsuccinate. At amembrane potential of 50 mV,K^Suc_0.5 is 102 ± 20 µM andK^Na+_0.5 is 22 ± 4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer andradiolabeled succinate uptake in hNaDC3-expressing oocytes indicate acharge-to-succinate ratio of 1:1 for the transport process, suggestinga Na⁺-to-succinate stoichiometry of 3:1. pH titration ofcitrate-induced currents shows that hNaDC3 accepts preferentially thedivalent anionic form of citrate as a substrate. Li⁺inhibits succinate-induced currents in the presence of Na⁺.Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The humanNaDC3 gene is located on chromosome20q12-13.1, as evidenced by fluorescent in situ hybridization. Thegene is >80 kbp long and consists of 13 exons and 12 introns.

     

Secretory modulation of basolateral membrane inwardly rectified K(+) channel in guinea pig distal colonic crypts

Cell-attached recordings revealedK⁺ channel activity in basolateral membranes ofguinea pig distal colonic crypts. Inwardly rectified currents wereapparent with a pipette solution containing 140 mM K⁺.Single-channel conductance () was 9 pS at the resting membrane potential. Another inward rectifier with  of 19 pS was observed occasionally. At a holding potential of 80 mV,  was 21 and 41 pS,respectively. Identity as K⁺ channels was confirmed afterpatch excision by changing the bath ion composition. From reversalpotentials, relative permeability of Na⁺ overK⁺ (P_Na/P_K)was 0.02 ± 0.02, withP_Rb/P_K = 1.1 andP_Cl/P_K < 0.03. Spontaneous open probability (P_o) of the 9-pSinward rectifier (^gpK_ir) was voltageindependent in cell-attached patches. Both a low(P_o = 0.09 ± 0.01) and a moderate(P_o = 0.41 ± 0.01) activity mode wereobserved. Excision moved ^gpK_ir to the mediumactivity mode; P_o of^gpK_ir was independent of bath Ca²⁺activity and bath acidification. Addition of Cl andK⁺ secretagogues altered P_o of^gpK_ir. Forskolin or carbachol (10 µM)activated the small-conductance ^gpK_ir inquiescent patches and increased P_o inlow-activity patches. K⁺ secretagogues, either epinephrine(5 µM) or prostaglandin E₂ (100 nM), decreasedP_o of ^gpK_ir in activepatches. This ^gpK_ir may be involved inelectrogenic secretion of Cl and K⁺ acrossthe colonic epithelium, which requires a large basolateral membraneK⁺ conductance during maximal Cl secretionand, presumably, a lower K⁺ conductance during primaryelectrogenic K⁺ secretion.

     

Age-dependent response of the electrocardiogram to K(+) channel blockers in mice

Developmental changes in electrocardiogram (ECG) andresponse to selective K⁺ channelblockers were assessed in conscious, unsedated neonatal (days 1, 7, 14) and adult male mice(>60 days of age). Mean sinus R-R interval decreased from 120 ± 3 ms in day 1 to 110 ± 3 ms inday 7, 97 ± 3 ms inday 14, and 81 ± 1 ms in adultmice (P < 0.001 by ANOVA; all 3 groups different from day 1). Inparallel, the mean P-R interval progressively decreased duringdevelopment. Similarly, the mean Q-T interval decreased from 62 ± 2 ms in day 1 to 50 ± 2 ms inday 7, 47 ± 8 ms inday 14 neonatal mice, and 46 ± 2 ms in adult mice (P < 0.001 byANOVA; all 3 groups are significantly different fromday 1).Q-T_c was calculated asQ- interval.Q-T_c significantly shortened from179 ± 4 ms in day 1 to 149 ± 5 ms in day 7 mice(P < 0.001). In addition, the J junction-S-T segment elevation observed in day1 neonatal mice resolved by day14. Dofetilide (0.5 mg/kg), the selective blocker ofthe rapid component of the delayed rectifier(I_Kr) abolished S-T segment elevation and prolonged Q-T andQ-T_c intervals in day 1 neonates but not in adult mice.In contrast, 4-aminopyridine (4-AP, 2.5 mg/kg) had no effect onday 1 neonates but in adults prolongedQ-T and Q-T_c intervals andspecifically decreased the amplitude of a transiently repolarizingwave, which appears as an r' wave at the end of the apparent QRSin adult mice. In conclusion, ECG intervals and configuration changeduring normal postnatal development in the mouse.K⁺ channel blockers affect themouse ECG differently depending on age. These data are consistent withthe previous findings that the dofetilide-sensitiveI_Kr is dominantin day 1 mice, whereas 4-AP-sensitivecurrents, the transiently repolarizingK⁺ current, and the rapidlyactivating, slowly inactivating K⁺current are the dominant K⁺currents in adult mice. This study provides background information useful for assessing abnormal development in transgenic mice.

     

Calcium dependence of C-type natriuretic peptide-formed fast K+ channel

The lipid bilayertechnique was used to characterize theCa²⁺ dependence of a fastK⁺ channel formed by a synthetic17-amino acid segment [OaCNP-39-(1-17)] ofa 39-amino acid C-type natriuretic peptide (OaCNP-39) found in platypus (Ornithorhynchusanatinus) venom (OaV). TheOaCNP-39-(1-17)-formed K⁺ channel was reversiblydependent on1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-buffered cis (cytoplasmic)Ca²⁺ concentration([Ca²⁺]_cis).The channel was fully active when[Ca²⁺]_ciswas >10⁴ M andtrans (luminal)Ca²⁺ concentration was 1.0 mM, butnot at low[Ca²⁺]_cis.The open probability of single channels increased from zero at1 × 10⁶ McisCa²⁺ to 0.73 ± 0.17 (n = 22) at10³ McisCa²⁺. Channel openings to themaximum conductance of 38 pS were rapidly and reversibly activated when[Ca²⁺]_cis,but not transCa²⁺ concentration(n = 5), was increased to >5 × 10⁴ M(n = 14). Channel openings to thesubmaximal conductance of 10.5 pS were dominant at5 × 10⁴ MCa²⁺.K⁺ channels did not open whencisMg²⁺ orSr²⁺ concentrations were increasedfrom zero to 10³ M or when[Ca²⁺]_ciswas maintained at 10⁶ M(n = 3 and 2). The Hill coefficientand the inhibition constant were 1 and 0.8 × 10⁴ McisCa²⁺, respectively. Thisdependence of the channel on high[Ca²⁺]_cissuggests that it may become active under1) physiological conditions whereCa²⁺ levels are high, e.g., duringcardiac and skeletal muscle contractions, and2) pathological conditions that leadto a Ca²⁺ overload, e.g., ischemicheart and muscle fatigue. The channel could modify a cascade ofphysiological functions that are dependent on theCa²⁺-activatedK⁺ channels, e.g., vasodilationand salt secretion.

     

Modulation of BK(Ca) channel activity by fatty acids: structural requirements and mechanism of action

To determinethe mechanism of fatty acid modulation of rabbit pulmonary arterylarge-conductance Ca²⁺-activated K⁺(BK_Ca) channel activity, we studied effects of fatty acidsand other lipids on channel activity in excised patches withpatch-clamp techniques. The structural features of the fatty acidrequired to increase BK_Ca channel activity (or averagenumber of open channels, NP_o) were identified tobe the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which havea sufficiently long alkyl chain (C  8), produced a decrease inNP_o. Neutral and short-chain lipids did notalter NP_o. Screening of membrane surface chargewith high-ionic-strength bathing solutions (330 mM K⁺ or130 mM K⁺, 300 mM Na⁺) did not alter themodulation of the BK_Ca channel NP_oby fatty acids and other charged lipids, indicating that channelmodulation is unlikely to be due to an alteration of the membraneelectric field or the attraction of local counterions to the channel.Fatty acids and other negatively charged lipids were able to modulate BK_Ca channel activity in bathing solutions containing 0 mMCa²⁺, 20 mM EGTA, suggesting that calcium is not requiredfor this modulation. Together, these results indicate that modulationof BK_Ca channels by fatty acids and other charged lipidsmost likely occurs by their direct interaction with the channel proteinitself or with some other channel-associated component.

     

Genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and colon

The action of the isoflavonegenistein on the cystic fibrosis transmembrane conductance regulator(CFTR) has been studied in many cell systems but not in intact murinetissues. We have investigated the action of genistein on murine tissuesfrom normal and cystic fibrosis (CF) mice. Genistein increased theshort-circuit current (I_sc) in tracheal(16.4 ± 2.8 µA/cm²) and colonic (40.0 ± 4.4 µA/cm²) epithelia of wild-type mice. This increase wasinhibited by furosemide, diphenylamine-2-carboxylate, andglibenclamide, but not by DIDS. In contrast, genistein produced nosignificant change in the I_sc of the trachealepithelium (0.9 ± 1.1 µA/cm²) and decreased theI_sc of colons from CF null (13.1 ± 2.3 µA/cm²) and F508 mice (10.3 ± 1.3 µA/cm²). Delivery of a human CFTRcDNA-liposome complex to the airways of CF null mice restored thegenistein response in the tracheas to wild-type levels. Tracheas fromF508 mice were also studied: 46% of trachea showed no response togenistein, whereas 54% gave an increase in I_scsimilar to that in wild type. We conclude that genistein activatesCFTR-mediated Cl secretion in the murine trachea anddistal colon.

     

Modulation of the sarcolemmal L-type current by alteration in SR Ca2+ release

Modulation of the L-type current by sarcoplasmicreticulum (SR) Ca²⁺ release hasbeen examined in patch-clamped mouse myotubes. Inhibition of SRCa²⁺ release by inclusion ofryanodine in the internal solution shifted the half-activating voltage(V_0.5) of theL-type current from 1.1 ± 2.1 to 7.7 ± 1.7 mV. Rutheniumred in the internal solution shiftedV_0.5 from 5.4 ± 1.9 to 3.2 ± 4.1 mV. Chelation of myoplasmic Ca²⁺ with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid perfusion shiftedV_0.5 from 4.4 ± 1.7 to 3.5 ± 3.3 mV and increased the peak current.Extracellular caffeine (1 mM), which should enhance SRCa²⁺ release, significantlydecreased the peak Ca²⁺ current.In low (0.1 mM) internal EGTA, myotube contraction was abolished byinternal perfusion with ryanodine or ruthenium red, whereas addition ofcaffeine to the extracellular solution lowered the contractilethreshold, indicating that these modulators of SRCa²⁺ release had the expectedeffects on contraction. Therefore, SR Ca²⁺ release appears to modulatethe sarcolemmal L-type current, suggesting a retrograde communicationfrom the SR to the sarcolemmal L-type channels inexcitation-contraction coupling.

     

Characteristics of L-lactic acid transport in basal membrane vesicles of human placental syncytiotrophoblast

Thecharacteristics of L-lactic acid transport across thetrophoblast basal membrane were investigated and compared with those across the brush-border membrane by using membrane vesicles isolated from human placenta. The uptake ofL-[¹⁴C]lactic acid into basal membranevesicles was Na⁺ independent, and an uphill transport wasobserved in the presence of a pH gradient([H⁺]_out > [H⁺]_in).L-[¹⁴C]lactic acid uptake exhibitedsaturation kinetics with a K_m value of 5.89 ± 0.68 mM in the presence of a pH gradient.p-Chloromercuribenzenesulfonate and-cyano-4-hydroxycinnamate inhibited the initial uptake, whereas phloretin or 4,4'-diisothiocyanostilbene-2,2'-disulfonate did not.Mono- and dicarboxylic acids suppressed the initial uptake. Inconclusion, L-lactic acid transport in the basal membraneis H⁺ dependent and Na⁺ independent, as is alsothe case for the brush-border membrane transport, and itscharacteristics resemble those of monocarboxylic acid transporters.However, there were several differences in the effects of inhibitorsbetween basal and brush-border membrane vesicles, suggesting that thetransporter(s) involved in L-lactic acid transport in thebasal membrane of placental trophoblast may differ from those in thebrush-border membrane.

     

Intermediate-conductance Ca2+-activated K+ channel is expressed in C2C12 myoblasts and is downregulated during myogenesis

We report here the expression in C₂C₁₂ myoblasts of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel. The IK_Ca current, recorded under perforated-patch configuration, had a transient time course when activated by ionomycin (0.5 µM; peak current density 26.2 ± 3.7 pA/pF; n = 10), but ionomycin (0.5 µM) + 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one (100 µM) evoked a stable outward current (28.4 ± 8.2 pA/pF; n = 11). The current was fully inhibited by charybdotoxin (200 nM), clotrimazole (2 µM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (300 µM), but not by tetraethylammonium (1 mM) or D-tubocurarine (300 µM). Congruent with the IK_Ca channel, elevation of intracellular Ca²⁺ in inside-out patches resulted in the activation of a voltage-insensitive K⁺ channel with weak inward rectification, a unitary conductance of 38 ± 6 pS (at negative voltages), and an IC₅₀ for Ca²⁺ of 530 nM. The IK_Ca channel was activated metabotropically by external application of ATP (100 µM), an intracellular Ca²⁺ mobilizer. Under current-clamp conditions, ATP application resulted in a membrane hyperpolarization of 35 mV. The IK_Ca current downregulated during myogenesis, ceasing to be detectable 4 days after the myoblasts were placed in differentiating medium. Downregulation was prevented by the myogenic suppressor agent basic FGF (bFGF). We also found that block of the IK_Ca channel by charybdotoxin did not inhibit bFGF-sustained myoblast proliferation. These observations show that in C₂C₁₂ myoblasts the IK_Ca channel expression correlates inversely with differentiation, yet it does not appear to have a role in myoblast proliferation. ATP; cell proliferation     

LY-294002-inhibitable PI 3-kinase and regulation of baseline rates of Na(+) transport in A6 epithelia

Blocker-inducednoise analysis of epithelial Na⁺ channels (ENaCs) was usedto investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na⁺transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na⁺ transport rates(I_Na) of 4.0 ± 0.1 (unstimulated) and9.1 ± 0.9 µA/cm² (aldosterone), 10 µM LY-294002caused, following a relatively small initial increase of transport, acompletely reversible inhibition of transport within 90 min to 33 ± 6% and 38 ± 2% of respective baseline values. Initialincreases of transport could be attributed to increases of channel openprobability (P_o) within 5 min to 143 ± 17% (unstimulated) and 142 ± 10% of control (aldosterone) frombaseline P_o averaging near 0.5. Inhibition oftransport was due to much slower decreases of functional channeldensities (N_T) to 28 ± 4% (unstimulated)and 35 ± 3% (aldosterone) of control at 90 min. LY-294002 (50 µM) caused larger but completely reversible increases ofP_o (215 ± 38% of control at 5 min) andmore rapid but only slightly larger decreases ofN_T. Basolateral exposure to LY-294002 induced nodetectable effect on transport, P_o or N_T. We conclude that an LY-294002-sensitive PI3-kinase plays an important role in regulation of transport bymodulating N_T and P_o ofENaCs, but only when presented to apical surfaces of the cells.

     

K+ channel KVLQT1 located in the basolateral membrane of distal colonic epithelium is not essential for activating Cl- secretion

The cellular mechanism for Cl^– and K⁺ secretion in the colonic epithelium requires K⁺ channels in the basolateral and apical membranes. Colonic mucosa from guinea pig and rat were fixed, sectioned, and then probed with antibodies to the K⁺ channel proteins K_VLQT1 (Kcnq1) and minK-related peptide 2 (MiRP2, Kcne3). Immunofluorescence labeling for Kcnq1 was most prominent in the lateral membrane of crypt cells in rat colon. The guinea pig distal colon had distinct lateral membrane immunoreactivity for Kcnq1 in crypt and surface cells. In addition, Kcne3, an auxiliary subunit for Kcnq1, was detected in the lateral membrane of crypt and surface cells in guinea pig distal colon. Transepithelial short-circuit current (I_sc) and transepithelial conductance (G_t) were measured for colonic mucosa during secretory activation by epinephrine (EPI), prostaglandin E₂ (PGE₂), and carbachol (CCh). HMR1556 (10 µM), an inhibitor of Kcnq1 channels (Gerlach U, Brendel J, Lang HJ, Paulus EF, Weidmann K, Brüggemann A, Busch A, Suessbrich H, Bleich M, and Greger R. J Med Chem 44: 3831–3837, 2001), partially (50%) inhibited Cl^– secretory I_sc and G_t activated by PGE₂ and CCh in rat colon with an IC₅₀ of 55 nM, but in guinea pig distal colon Cl^– secretory I_sc and G_t were unaltered. EPI-activated K⁺-secretory I_sc and G_t also were essentially unaltered by HMR1556 in both rat and guinea pig colon. Although immunofluorescence labeling with a Kcnq1 antibody supported the basolateral membrane presence in colonic epithelium of the guinea pig as well as the rat, the Kcnq1 K⁺ channel is not an essential component for producing Cl^– secretion. Other K⁺ channels present in the basolateral membrane presumably must also contribute directly to the K⁺ conductance necessary for K⁺ exit during activation of Cl^– secretion in the colonic mucosa. HMR1556; K⁺ secretion; epinephrine; prostaglandin E₂; cholinergic     

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na⁺ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(R_T) are 5.9 mV and 829  · cm², respectively. The apical membranepotential (V_A) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (R_A/R_B) is 2.8. Apicalamiloride hyperpolarized V_A by 19.7 mV andtripled R_A/R_B. AcAMP-elevating cocktail depolarized V_A by 17.6 mV, decreased R_A/R_B by60%, increased short-circuit current by 6 µA/cm²,decreased R_T by 155  · cm², and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na⁺ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm² · h¹). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa⁺ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na⁺] and [Cl].

     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia

It has been reported thatsecretory mammary epithelial cells (MEC) release ATP, UTP, and UDP uponmechanical stimulation. Here we examined the physiological changescaused by ATP/UTP in nontransformed, clonal mouse mammary epithelia(31EG4 cells). In control conditions, transepithelial potential (apicalside negative) and resistance were 4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4  · cm², respectively. The apicalmembrane potential was 43.9 ± 1.7 mV, and the ratio of apicalto basolateral membrane resistance (R_A/R_B) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranescaused large voltage and resistance changes with an EC₅₀ of~24 µM (apical) and ~30 µM (basal). Apical ATP/UTP (100 µM)depolarized apical membrane potential by 17.6 ± 0.8 mV (n = 7) and decreasedR_A/R_B by a factor of3. The addition of adenosine to either side (100 µM) hadno effect on any of these parameters. The ATP/UTP responses werepartially inhibited by DIDS and suramin and mediated by a transientincrease in free intracellular Ca²⁺ concentration (427 ± 206 nM; 15-25 µM ATP, apical; n = 6). This Ca²⁺ increase was blocked by cyclopiazonic acid, by BAPTA,or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbedfluid in the resting state, and ATP or UTP increased fluid secretion by5.6 ± 3 µl · cm² · h¹(n = 10). Pharmacology experiments indicate that 31EG4epithelia contain P2Y₂ purinoceptors on the apical andbasolateral membranes, which upon activation stimulate apicalCa²⁺-dependent Cl channels and cause fluid secretion acrossthe monolayer. This suggests that extracellular nucleotides could playa fundamental role in mammary gland paracrine signaling and theregulation of milk composition in vivo.

     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.