Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells

The hypothesis that protein kinase C (PKC) isable to regulate the whole cell Ca-activated K(K_Ca) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog1,2-dioctanoyl-sn-glycerol (DOG) at 10 µM attenuated the current-voltage(I-V)relationship of the K_Ca current significantly and reduced the K_Cacurrent at +70 mV by 70 ± 4% (n = 14). In contrast, 10 µM DOG after pretreatment of the cells with 1 µM calphostin C or 1 µM PKC inhibitor peptide, selective PKCinhibitors, and 10 µM1,3-dioctanoyl-sn-glycerol, aninactive diacylglycerol analog, did not significantly alter theK_Ca current. Furthermore, thecatalytic subunit of PKC (PKC_C)at 0.1 U/ml attenuated theI-Vrelationship of the K_Ca currentsignificantly, reduced the K_Cacurrent at +70 mV by 44 ± 3% (n = 17), and inhibited the activity of singleK_Ca channels at 0 mV by 79 ± 9% (n = 6). In contrast, 0.1 U/mlheat-inactivated PKC_C did notsignificantly alter the K_Cacurrent or the activity of singleK_Ca channels. Thus these resultssuggest that PKC is able to considerably attenuate theK_Ca current of freshly isolatedrat tail artery smooth muscle cells independently of effects of PKC onlocal Ca release events, most likely by a direct effect on theK_Ca channel.     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Letters to the Editor

The following is the abstract of the article discussed in thesubsequent letter:

Mitchell, Claire H., Jin Jun Zhang, Liwei Wang, andTim J. C. Jacob. Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases. Am. J. Physiol. 272 (Cell Physiol. 41): C212-C222, 1997.Thewhole cell recording technique was used to examine an outwardlyrectifying chloride current activated by hypotonic shock in bovinepigmented ciliary epithelial (PCE) cells. Removal of internal andexternal Ca²⁺ did not affect the activation of thesecurrents, but they were abolished by the phospholipase C inhibitorneomycin. The current was blocked by5-nitro-2-(3-phenylpropylamino)benzoic acid,4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) in avoltage-dependent manner, but tamoxifen, dideoxyforskolin, andquinidine did not affect it. This blocking profile differs from that ofthe volume-sensitive chloride channel in neighboring nonpigmentedciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob. J. Physiol. Lond. 491: 743-755, 1996), and thisdifference implies that the volume responses of the two cell types aremediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTPS) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent blockof the GTPS-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells incalphostin C, an inhibitor of protein kinase C (PKC). Removal ofCa²⁺ did not affect activation of the current by GTPSbut extended the duration of the response. Inhibition of phospholipaseA₂ (PLA₂) with p-bromophenacyl bromideprevented the activation of the hypotonically induced current and alsoinhibited the current once activated by hypotonic solution. Thefindings imply that the hypotonic response in PCE cells is mediated byboth phospholipase C (PLC) and PLA₂. Both phospholipasesgenerate arachidonic acid, and, in addition, the PLC pathway regulatesthe PLA₂ pathway via a PKC-dependent phosphorylation ofPLA₂.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Characterization of a novel voltage-dependent outwardly rectifying anion current in Caenorhabditis elegans oocytes

An inwardly rectifying swelling- and meiotic cell cycle-regulated anion current carried by the ClC channel splice variant CLH-3b dominates the whole cell conductance of the Caenorhabditis elegans oocyte. Oocytes also express a novel outwardly rectifying anion current termed I_Cl,OR. We recently identified a worm strain carrying a null allele of the clh-3 gene and utilized oocytes from these animals to characterize I_Cl,OR biophysical properties. The I_Cl,OR channel is strongly voltage dependent. Outward rectification is due to voltage-dependent current activation at depolarized voltages and rapid inactivation at voltages more hyperpolarized than approximately +20 mV. Apparent channel open probability is zero at voltages less than +20 mV. The channel has a 4:1 selectivity for Cl^– over Na⁺ and an anion selectivity sequence of SCN^– > I^– > Br^– > Cl^– > F^–. I_Cl,OR is relatively insensitive to most conventional anion channel inhibitors including DIDS, 4,4'-dinitrostilbene-2,2'-disulfonic acid, 9-anthracenecarboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid. However, the current is rapidly inhibited by niflumic acid, metal cations including Gd³⁺, Cd²⁺, and Zn²⁺, and bath acidification. The combined biophysical properties of I_Cl,OR are distinct from those of other anion currents that have been described. During oocyte meiotic maturation, I_Cl,OR activity is rapidly downregulated, suggesting that the channel may play a role in oocyte Cl^– homeostasis, development, cell cycle control, and/or ovulation. chloride channel; ovulation; cell cycle; meiotic maturation     

Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes

It is generally accepted that gases such asCO₂ cross cell membranes bydissolving in the membrane lipid. No role for channels or pores in gastransport has ever been demonstrated. Here we ask whether expression ofthe water channel aquaporin-1 (AQP1) enhances theCO₂ permeability ofXenopus oocytes. We expressed AQP1 inXenopus oocytes by injecting AQP1cRNA, and we assessed CO₂permeability by using microelectrodes to monitor the changes inintracellular pH (pH_i) producedby adding 1.5% CO₂/10 mM to (or removing it from) theextracellular solution. Oocytes normally have an undetectably low levelof carbonic anhydrase (CA), which eliminates theCO₂ hydration reaction as arate-limiting step. We found that expressing AQP1 (vs. injectingwater) had no measurable effect on the rate ofCO₂-inducedpH_i changes in such low-CAoocytes: adding CO₂ causedpH_i to fall at a mean initial rateof 11.3 × 10⁴ pHunits/s in control oocytes and 13.3 × 10⁴ pH units/s in oocytesexpressing AQP1. When we injected oocytes with water, and a few dayslater with CA, the CO₂-inducedpH_i changes in these water/CAoocytes were more than fourfold faster than in water-injected oocytes(acidification rate, 53 × 10⁴ pH units/s).Ethoxzolamide (ETX; 10 µM), a membrane-permeant CA inhibitor, greatlyslowed the pH_i changes (16.5 × 10⁴ pHunits/s). When we injected oocytes with AQP1 cRNA and then CA, theCO₂-inducedpH_i changes in these AQP1/CAoocytes were ~40% faster than in the water/CA oocytes (75 × 10⁴ pH units/s), and ETXreduced the rates substantially (14.7 × 10⁴ pH units/s). Thus, inthe presence of CA, AQP1 expression significantly increases theCO₂ permeability of oocytemembranes. Possible explanations include1) AQP1 expression alters the lipidcomposition of the cell membrane, 2)AQP1 expression causes overexpression of a native gas channel,and/or 3) AQP1 acts as achannel through which CO₂ canpermeate. Even if AQP1 should mediate aCO₂ flux, it would remain to bedetermined whether this CO₂movement is quantitatively important.

     

Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1or its C189S mutant

A recent study onXenopus oocytes [N. L. Nakhoul,M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (CellPhysiol. 43): C543-548, 1998] injected withcarbonic anhydrase showed that expressing aquaporin 1 (AQP1) increasesby ~40% the rate at which exposing the cell toCO₂ causes intracellular pH tofall. This observation is consistent with several interpretations.Overexpressing AQP1 might increase apparentCO₂ permeability by1) allowingCO₂ to pass through AQP1,2) stimulating injected carbonicanhydrase, 3) enhancing theCO₂ solubility of the membrane'slipid, or 4) increasing theexpression of a native "gas channel." The purpose of the presentstudy was to distinguish among these possibilities. We found thatexpressing the H₂O channel AQP1 inXenopus oocytes increases theCO₂ permeability of oocytes in anexpression-dependent fashion, whereas expressing theK⁺ channel ROMK1 has no effect.The mercury derivativep-chloromercuriphenylsulfonic acid(PCMBS), which inhibits the H₂Omovement through AQP1, also blocks the AQP1-dependent increase inCO₂ permeability. Themercury-insensitive C189S mutant of AQP1 increases theCO₂ permeability of the oocyte tothe same extent as does the wild-type channel. However, the C189S-dependent increase in CO₂permeability is unaffected by treatment with PCMBS. These data rule outoptions 2-4 listed above. Thusour results suggest that CO₂passes through the pore of AQP1 and are the first data to demonstratethat a gas can enter a cell by a means other than diffusing through themembrane lipid.

     

Dynamics of calcium regulation of chloride currents in Xenopus oocytes

Ca-activated Cl currents are widely expressed in many cell typesand play diverse and important physiological roles. TheXenopus oocyte is a good model systemfor studying the regulation of these currents. We previously showedthat inositol 1,4,5-trisphosphate (IP₃) injection intoXenopus oocytes rapidly elicits anoninactivating outward Cl current(I_Cl1-S)followed several minutes later by the development of slow inward(I_Cl2) andtransient outward(I_Cl1-T) Clcurrents. In this paper, we investigate whether these three currentsare mediated by the same or different Cl channels. Outward Cl currentswere more sensitive to Ca than inward Cl currents, as shown byinjection of different amounts of Ca or by Ca influx through aheterologously expressed ligand-gated Ca channel, the ionotropicglutamate receptor iGluR3. These data could be explained by twochannels with different Ca affinities or one channel with a higher Caaffinity at depolarized potentials. To distinguish between thesepossibilities, we determined the anion selectivity of the threecurrents. The anion selectivity sequences for the three currents werethe same (I > Br > Cl), butI_Cl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, afterconsideration of possible errors, the hypothesis that a single type ofCl channel underlies the complex waveforms of the three differentmacroscopic Ca-activated Cl currents inXenopus oocytes remains a viable alternative.

     

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation

We have designed and synthesizedbenzo[c]quinolizinium derivatives and evaluated their effects on theactivity of G551D cystic fibrosis transmembrane conductance regulator(CFTR) expressed in Chinese hamster ovary and Fisher ratthyroid cells. We demonstrated, using iodide efflux, whole cell patchclamp, and short-circuit recordings, that5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91)restored the activity of G551D CFTR (EC₅₀ = 85 µM)and activated CFTR in Calu-3 cells (EC₅₀ = 47 µM).MPB-91 has no effect on the ATPase activity of wild-type and G551DNBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylatekinase activities of purified NBD2. The activation of CFTR by MPB-91 isindependent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTRhaving 10 mutated protein kinase A sites (10SA-CFTR). The newpharmacological agent MPB-91 may be an important candidate drug toameliorate the ion transport defect associated with CF and to point outa new pathway to modulate CFTR activity.

     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells

Rapamycin and FK-506 are immunosuppressive drugs thatbind a ubiquitous immunophilin, FKBP12, but immunosuppressivemechanisms and side effects appear to be different. Rapamycin bindsrenal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na⁺ channels in A6cells. Channel NP_o was 0.5 ± 0.08 (n = 6)during the first 5 min but fell close to zero after 20 min. Application of 1 µM rapamycin reactivated Na⁺ channels(NP_o = 0.47 ± 0.1; n=6), but 1 µMFK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor,mimicked the rapamycin-induced reactivation in a nonadditive manner.However, rapamycin did not reactivate Na⁺ channels if cellswere exposed to 1 µM FK-506 before rapamycin. In PKC assays,rapamycin was as effective as the PKC inhibitor; however, epithelialNa⁺ channel (ENaC) phosphorylation was low under baselineconditions and was not altered by PKC inhibitors or activators. Theseresults suggest that rapamycin activates Na⁺ channels bybinding FKBP12 and inhibiting PKC, and, in renal cells, despite bindingthe same immunophilin, rapamycin and FK-506 activate differentintracellular signaling pathways.

     

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.

     

Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2

Activity of the human (h) cystic fibrosis transmembrane conductance regulator (CFTR) channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus (X)CFTR is more responsive to PKC than PKA stimulation. We investigated the interaction between the two kinases in XCFTR. We expressed XCFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of activation after PKC stimulation was about eightfold that without pretreatment with PKC agonist. hCFTR, expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied single-channel properties and found that the single-channel open probability (P_o) with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC preactivation and that single-channel conductance () was increased by 22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH₂-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in P_o (with a small change in ) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains. cystic fibrosis; chloride channel; protein kinases; ATP binding cassette proteins     

Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia

Activation ofprotein kinase C with phorbol 12-myristate 13-acetate (PMA) causedcomplex transient perturbations of amiloride-sensitive short-circuitNa⁺ currents(I_Na) in A6epithelia and frog skins that were tissue and concentration dependent.A noninvasive channel blocker pulse method of noise analysis (18) wasused to investigate how PMA caused time-dependent changes of apicalmembrane epithelial Na⁺ channel(ENaC) single-channel currents, channel open probabilities (P_o), andchannel densities(N_T). In A6epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependentsustained decreases ofP_o (~55% belowcontrol, 50 nM) and rapid compensatory transient increases ofN_T within 7 min(~220% above control, 50 nM), resulting in either small transientincreases of I_Naat 5 nM PMA or small biphasic decreases ofI_Na at 50 nM PMA.In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin causedafter a delay of at least 10 min transient increases ofN_T to~60-70% above control at 30-60 min. Unlike A6 epithelia,P_o was increased~15% above control within 7 min and remained within±10-15% of control for the duration of the 2-h experiments.Despite differences in the time courses of secondary inhibition oftransport in A6 epithelia and frog skin, the delayed downregulation oftransport was due to time-dependent decreases ofN_T from theirpreelevated levels in both tissues. WhereasP_o is decreasedwithin minutes in A6 epithelia as measured by noise analysis or bypatch clamp (8), the discrepancy in regulation ofN_T in A6epithelia as measured by noise analysis and patch clamp is most likelyexplained by the inability of on-cell patches formed before treatmentof tissues with PMA to respond to regulation of their channeldensities.

     

Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport

We report, for the epithelialNa⁺ channel (ENaC) in A6 cells,the modulation by cell pH (pH_c)of the transepithelial Na⁺ current(I_Na), thecurrent through the individual Na⁺channel (i), the openNa⁺ channel density(N_o), and thekinetic parameters of the relationship betweenI_Na and theapical Na⁺ concentration. Thei andN_o were evaluatedfrom the Lorentzian I_Na noise inducedby the apical Na⁺ channel blocker6-chloro-3,5-diaminopyrazine-2-carboxamide.pH_c shifts were induced, understrict and volume-controlled experimental conditions, byapical/basolateral NH₄Cl pulses orbasolateral arrest of theNa⁺/H⁺exchanger (Na⁺ removal; block byethylisopropylamiloride) and were measured with the pH-sensitive probe2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Thechanges in pH_c were positivelycorrelated to changes inI_Na and theapically dominated transepithelial conductance. The sole pH_c-sensitive parameter underlyingI_Na wasN_o. Only thesaturation value of theI_Na kinetics wassubject to changes in pH_c.pH_c-dependent changes inN_o may be causedby influencingP_o, the ENaC openprobability, or/and the total channel number,N_T = N_o/P_o.

     

Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes

We recently identified a cDNA clone frommouse small intestine, which appears to be involved in folate transportwhen expressed in Xenopus oocytes. Theopen reading frame of this clone is identical to that of the reducedfolate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley,and K. H. Cowan. J. Biol. Chem. 269: 17-20,1994). The characteristics of this cDNA clone [previously referred toas intestinal folate carrier 1 (IFC-1)] expressed inXenopus oocytes, however, were foundto be different from the characteristics of folate transport in nativesmall intestinal epithelial cells. To further study these differences,we determined the characteristics of RFC when expressed in anintestinal epithelial cell line, IEC-6, and compared the findings toits characteristics when expressed inXenopus oocytes. RFC was stablytransfected into IEC-6 cells by electroporation; its cRNA wasmicroinjected into Xenopus oocytes.Northern blot analysis of poly(A)⁺RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFCwas found to be fourfold higher than uptake in control sublines. Thisincrease in folic acid and 5-MTHF uptake was inhibited by treatingIEC-6/RFC cells with cholesterol-modified antisense DNAoligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity(V_max) of theuptake process [the apparent Michaelis-Menten constant(K_m) alsochanged (range was 0.31 to 1.56 µM), but no specific trend wasseen]. In both IEC-6/RFC and control sublines, the uptake of bothfolic acid and 5-MTHF displayed 1)pH dependency, with a higher uptake at acidic pH 5.5 compared with pH7.5, and 2) inhibition to the sameextent by both reduced and oxidized folate derivatives. Thesecharacteristics are very similar to those seen in native intestinalepithelial cells. In contrast, RFC expressed inXenopus oocytes showed1) higher uptake at neutral andalkaline pH 7.5 compared with acidic pH 5.5 and2) higher sensitivity to reducedcompared with oxidized folate derivatives. Results of these studiesdemonstrate that the characteristics of RFC vary depending on the cellsystem in which it is expressed. Furthermore, the results may suggestthe involvement of cell- or tissue-specific posttranslationalmodification(s) and/or the existence of an auxiliary proteinthat may account for the differences in the characteristics of theintestinal RFC when expressed inXenopus oocytes compared with whenexpressed in intestinal epithelial cells.

     

Neuronal swelling and surface area regulation: elevated intracellular calcium is not a requirement

Neurons aremechanically robust. During prolonged swelling, molluscan neurons cantriple their apparent membrane area. They gain surface area andcapacitance independent of extracellular Ca concentration([Ca]_e), but it isunknown if an increase in intracellular Ca concentration([Ca]_i) isnecessary. If Ca for stimulating exocytosis is unnecessary, it ispossible that swelling-induced membrane tension changes directlytrigger surface area readjustments. If, however, Ca-mediated but nottension-mediated membrane recruitment is responsible for surface areaincreases, swelling neurons should sustain elevated levels of[Ca]_i. The purpose ofthis investigation is to determine if the[Ca]_i in swellingneurons attains levels high enough to promote exocytosis and if anysuch increase is required. Lymnaeaneurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethyleneglycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 µM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min)were done at either a standard[Ca]_e or very low[Ca]_e level (0.9 mM or0.13 µM, respectively). In neither case did the[Ca]_i increase tolevels that drive exocytosis. We also monitored osmomechanically drivenmembrane dynamics [swelling, then formation and reversal ofvacuole-like dilations (VLDs)] with the[Ca]_i clamped below 40 nM via1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]_idid not change with swelling, and VLD behavior was unaffected,consistent with tension-driven,[Ca]_i-independent surface area adjustments. In addition, neurons with[Ca]_i clamped at 0.1 µM via an ionophore could produce VLDs. We conclude that, undermechanical stress, neuronal membranes are compliant by virtue ofsurface area regulatory adjustments that operate independent of[Ca]_i. The findingssupport the hypothesis that plasma membrane area is regulated in partby membrane tension.

     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

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.

     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa⁺ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na⁺ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa⁺ current (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.