Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations

Effects of basolateral monovalent cation replacements(Na⁺ byLi⁺,K⁺,Cs⁺, methylammonium, andguanidinium) on permeability to⁸⁶Rb of volume-sensitive cationchannels (VSCC) in the basolateral membrane and on regulatory volumedecrease (RVD), elicited by a hyposmotic shock, were studied in A6epithelia in the absence of apicalNa⁺ uptake. A complete and quickRVD occurred only when the cells were perfused withNa⁺ orLi⁺ saline. With both cations,hypotonicity increased basolateral ⁸⁶Rb release(R^bl_Rb), which reached a maximum after 15 min and declined back to control level. When the major cation wasK⁺,Cs⁺, methylammonium, orguanidinium, the RVD was abolished. Methylammonium induced a biphasictime course of cell thickness(T_c), with an initial decline ofT_c followed by a gradual increase.With K⁺,Cs⁺, or guanidinium,T_c increased monotonously afterthe rapid initial rise evoked by the hypotonic challenge. In thepresence of K⁺,Cs⁺, or methylammonium,R^bl_Rb remained high during most of thehypotonic period, whereas with guanidinium blockage of R^bl_Rb was initiated after 6 min ofhypotonicity, suggesting an intracellular location of the site ofaction. With all cations, 0.5 mM basolateralGd³⁺ completely blocked RVD andfully abolished the R^bl_Rb increaseinduced by the hypotonic shock. The lanthanide also blocked theadditional volume increase induced byCs⁺,K⁺, guanidinium, ormethylammonium. When pH was lowered from 7.4 to 6.0, RVD andR^bl_Rb were markedly inhibited. This studydemonstrates that the VSCCs in the basolateral membrane of A6 cells arepermeable to K⁺,Rb⁺,Cs⁺, methylammonium, andguanidinium, whereas a marked inhibitory effect is exerted byGd³⁺, protons, and possiblyintracellular guanidinium.

     

Dependence of Na+-K+ pump current-voltage relationship on intracellular Na+, K+, and Cs+ in rabbit cardiac myocytes

To examine effects of cytosolicNa⁺, K⁺, and Cs⁺ on the voltagedependence of the Na⁺-K⁺ pump, we measuredNa⁺-K⁺ pump current (I_p)of ventricular myocytes voltage-clamped at potentials(V_m) from 100 to +60 mV. Superfusates weredesigned to eliminate voltage dependence at extracellular pump sites.The cytosolic compartment of myocytes was perfused with patch pipette solutions with a Na⁺ concentration ([Na]_pip)of 80 mM and a K⁺ concentration from 0 to 80 mM or withsolutions containing Na⁺ in concentrations from 0.1 to 100 mM and K⁺ in a concentration of either 0 or 80 mM. When[Na]_pip was 80 mM, K⁺ in pipette solutionshad a voltage-dependent inhibitory effect on I_pand induced a negative slope of theI_p-V_m relationship. Cs⁺ in pipette solutions had an effect onI_p qualitatively similar to that ofK⁺. Increases in I_p with increasesin [Na]_pip were voltage dependent. The dielectriccoefficient derived from[Na]_pip-I_p relationships at thedifferent test potentials was 0.15 when pipette solutions included 80 mM K⁺ and 0.06 when pipette solutions were K⁺ free.

     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects ofendurance run training onNa⁺-dependentCa²⁺ regulation in rat leftventricular myocytes were examined. Myocytes were isolated fromsedentary and trained rats and loaded with fura 2. Contractile dynamicsand fluorescence ratio transients were recorded during electricalpacing at 0.5 Hz, 2 mM extracellular Ca²⁺ concentration, and 29°C.Resting and peak cytosolic Ca²⁺concentration([Ca²⁺]_c)did not change with exercise training. However, resting and peak[Ca²⁺]_cincreased significantly in both groups during 5 min of continuous pacing, although diastolic[Ca²⁺]_cin the trained group was less susceptible to this elevation ofintracellular Ca²⁺. Run trainingalso significantly reduced the rate of[Ca²⁺]_cdecay during relaxation. Myocytes were then exposed to 10 mM caffeinein the absence of external Na⁺ orCa²⁺ to trigger sarcoplasmicreticular Ca²⁺ release and tosuppress cellular Ca²⁺ efflux.This maneuver elicited an elevated steady-state[Ca²⁺]_c.External Na⁺ was then added, andthe rate of[Ca²⁺]_cclearance was determined. Run training significantly reduced the rateof Na⁺-dependent clearance of[Ca²⁺]_cduring the caffeine-induced contractures. These data demonstrate thatthe removal of cytosolic Ca²⁺ wasdepressed with exercise training under these experimental conditionsand may be specifically reflective of a training-induced decrease inthe rate of cytosolic Ca²⁺ removalviaNa⁺/Ca²⁺exchange and/or in the amount ofCa²⁺ moved across the sarcolemmaduring a contraction.     

Loss of cell volume regulation during metabolic inhibition in renal epithelial cells (A6): role of intracellular pH

In renalischemia, tubular obstruction induced by swelling of epithelialcells might be an important mechanism for reduction of the glomerularfiltration rate. We investigated ischemic cell swelling byexamining volume regulation of A6 cells during metabolic inhibition(MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume weremonitored by recording cell thickness (T_c). Intracellular pH (pH_c) measurements were performed with thepH-sensitive probe 5-chloromethyl-fluoresceine diacetate.T_c measurements showed that MI increases cellvolume. Cell swelling during MI is proportional to the rate ofNa⁺ transport and is not followed by a volume regulatoryresponse. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. Atransient acidification induced by a NH₄Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On theother hand, acute lowering of external pH to 5, simultaneously with thehypotonic shock, allowed the onset of RVD. However, this RVD wascompletely arrested ~10 min after the initiation of the hyposmoticchallenge. The inhibition of RVD appears to be related to thepronounced acidification that occurred within this time period. Incontrast, when external pH was lowered 20 min before the hyposmoticshock, RVD was absent. These data suggest that internal acidificationinhibits cellular volume regulation in A6 cells. Therefore, theintracellular acidification associated with MI might at least partlyaccount for the failure of volume regulation in swollen epithelial cells.

     

Red blood cells do not contribute to removal of K+ released from exhaustively working forearm muscle

K⁺ released from exercisingmuscle via K⁺ channels needs to beremoved from the interstitium into the blood to maintain high musclecell membrane potential and allow normal muscle contractility. Uptakeby red blood cells has been discussed as one mechanism that would alsoserve to regulate red blood cell volume, which was found to be constantdespite increased plasma osmolality and K⁺ concentration([K⁺_pl]). We evaluatedexercise-related changes in[K⁺_pl], pH, osmolality, meancellular Hb concentration, cell water, and red blood cellK⁺ concentration during exhaustivehandgrip exercise. Unidirectional ⁸⁶Rb⁺(K⁺) uptake by red blood cellswas measured in media with elevated extracellularK⁺, osmolarity, andcatecholamines to simulate particularly those exercise-related changesin plasma composition that are known to stimulateK⁺ uptake. During exercise[K⁺_pl] increased from 4.4 ± 0.7 to 7.1 ± 0.5 mmol/l plasma water and red blood cell K⁺ concentration increased from137.2 ± 6.0 to 144.6 ± 4.6 mmol/l cell water(P  0.05), but the intracellularK⁺-to-mean cellularHb concentration ratio did not change.⁸⁶Rb⁺uptake by red blood cells was increased by ~20% on stimulation, caused by activation of theNa⁺-K⁺pump andNa⁺-K⁺-2Clcotransport. Results indicate theK⁺ content of red blood cells didnot change as cells passed the exhaustively exercising forearm muscledespite the elevated [K⁺_pl]. The tendency for an increase in intracellularK⁺ concentration was due to aslight, although statistically not significant, decrease in red bloodcell volume. K⁺ uptake, althoughelevated, was too small to move significant amounts ofK⁺ into red blood cells. Ourresults suggest that red blood cells do not contribute to the removalof K⁺ released from muscle and donot regulate their volume by K⁺uptake during exhaustive forearm exercise.

     

Measurements of mitochondrial K+ fluxes in whole rat hearts using 87Rb-NMR

The rubidium efflux from hypothermic rat hearts perfused by theLangendorff method at 20°C was studied. At thistemperature ⁸⁷Rb-NMR efflux experiments showed theexistence of two ⁸⁷Rb pools: cytoplasmic and mitochondrial.Rat heart mitochondria showed a very slow exchange of mitochondrialRb⁺ for cytoplasmic K⁺. After washout ofcytosolic Rb⁺, mitochondria kept a stable Rb⁺level for >30 min. Rb⁺ efflux from mitochondria wasstimulated with 0.1 mM 2,4-dinitrophenol (DNP), by sarcolemmalpermeabilization and concomitant cellular energy depletion by saponin(0.01 mg/ml for 4 min) in the presence of a perfusate mimickingintracellular conditions, or by ATP-sensitive K (K_ATP)channel openers. DNP, a mitochondrial uncoupler, caused the onset ofmitochondrial Rb⁺ exchange; however, the washout was notcomplete (80 vs. 56% in control). Energy deprivation by saponin, whichpermeabilizes the sarcolemma, resulted in a rapid and completeRb⁺ efflux. The mitochondrial Rb⁺ efflux rateconstant (k) decreased in the presence of glibenclamide, aK_ATP channel inhibitor (5 µM;k = 0.204 ± 0.065 min¹; n = 8),or in the presence of ATP plus phosphocreatine (1.0 and 5.0 mM,respectively; k = 0.134 ± 0.021 min¹;n = 4) in the saponin experiments (saponin only;k = 0.321 ± 0.079 min¹; n = 3),indicating the inhibition of mitochondrial K_ATP channels. Thus hypothermia in combination with ⁸⁷Rb-NMR allowed theprobing of the mitochondrial K⁺ pool in whole heartswithout mitochondrial isolation.

     

Preservation of murine embryos in a state of dormancy at 4C

With the aim ofimproving preservation of blood products and organs fortransplantation, we designed solutions to induce a state of dormancy incells and tissues at 4°C. The solutions were devoid of combinationsof ions (e.g., K⁺,Rb⁺,Cs⁺, andNH⁺₄ withHCO₃,H₂PO₄, andCl) that are believed tobreak down low-density water in the entrance compartments of ionchannels, resulting in cyclical open states (normal water) and closedstates (low-density water). The total osmolality was always0.29-0.3 osmol/kgH₂O, made upof combinations of a di- or trisaccharide, a compatible solute, sodiumsulfate, citrate, or chloride, and 1.75 mMCaCl₂. The end point was the ability of murine embryos to progress to hatching in culture after preservation in such a solution at 4°C. Embryos hatched after 5 or6 days in some preservative solutions compared with 1-3 days inmost saline solutions; survival was improved by pretreatment withsodium butyrate.     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.

     

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse

The myoplasmic free Ca²⁺concentration([Ca²⁺]_i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca²⁺ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na⁺ was reducedfrom 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-modeNa⁺/Ca²⁺exchange (Ca²⁺ entry in exchangefor Na⁺ leaving the cell). Undernormal resting conditions, application oflow-Na⁺ solution only increased[Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting[Ca²⁺]_iof 42 nM. In other fibers,[Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca²⁺ release with caffeine (10 mM)and by inhibiting SR Ca²⁺ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa⁺/Ca²⁺exchange (Ca²⁺ removal from thecell in exchange for Na⁺ influx).These two agents caused a large increase in[Ca²⁺]_i,which then declined to a plateau level approximately twice the baseline[Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa²⁺ had been removed, theincrease in[Ca²⁺]_iduring the second exposure was very low, suggesting thatCa²⁺ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na⁺ solution produced a large,sustained increase in[Ca²⁺]_iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na⁺ solution in whichCa²⁺ had been removed, a sustainedincrease in[Ca²⁺]_iwas not observed, although[Ca²⁺]_iremained higher and declined slower than in normalNa⁺ solution. This suggests thatforward-modeNa⁺/Ca²⁺exchange contributed to the fall of[Ca²⁺]_iin normal Na⁺ solution, but whenextracellular Na⁺ was low, aprolonged elevation of[Ca²⁺]_icould activate reverse-modeNa⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess aNa⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.

     

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Cytoplasmic concentration of Mg²⁺([Mg²⁺]_i) was measured with a fluorescentindicator furaptra in ventricular myocytes enzymatically dissociatedfrom rat hearts (25°C). To study Mg²⁺ transport acrossthe cell membrane, cells were treated with ionomycin inCa²⁺-free (0.1 mM EGTA) and high-Mg²⁺ (10 mM)conditions to facilitate passive Mg²⁺ influx. Rate of riseof [Mg²⁺]_i due to the net Mg²⁺influx was significantly smaller in the presence of 130 mMextracellular Na⁺ than in its absence. We also tested theextracellular Na⁺ dependence of the net Mg²⁺efflux from cells loaded with Mg²⁺. After[Mg²⁺]_i was raised by ionomycin and highMg²⁺ to the level 0.5-0.6 mM above the basal value(~0.7 mM), washout of ionomycin and lowering extracellular[Mg²⁺] to 1.2 mM caused rapid decline of[Mg²⁺]_i in the presence of 140 mMNa⁺. This net efflux of Mg²⁺ was completelyinhibited by withdrawal of extracellular Na⁺ and waslargely attenuated by imipramine, a known inhibitor of Na⁺/Mg²⁺ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg²⁺ efflux andextracellular Na⁺ concentration([Na⁺]_o) had a Hill coefficient of 2 and[Na⁺]_o at half-maximal rate of 82 mM. Theseresults demonstrate the presence of Na⁺ gradient-dependentMg²⁺ transport, which is consistent withNa⁺/Mg²⁺ exchange, in cardiac myocytes.

     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

Examination of analytical methods for ions and saccharides in the extract of Phaseolus pulvini

With slight modifications, conventional assay procedures forK⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, NO₃^–, H₂PO₄^–, fructoseand fructose-yielding saccharides, and glucose were applicableto the extract of Phaseolus pulvini. About 10 ml of a hot-waterextract from about 30 mg fresh weight of the pulvini was sufficientfor separate measurement of the ions and saccharides named above. (Received August 7, 1979; )     

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.

     

Effect of osmolarity on LDL binding and internalization in hepatocytes

The present study has been performed to elucidate a possiblerole of cell volume in low-density lipoprotein (LDL) binding andinternalization (LDL_b+i). Asshown previously, increase of extracellular osmolarity (OSMe) andK⁺ depletion, both known to shrinkcells, interfere with the formation of clathrin-coated pits and thuswith LDL_b+i. On the other hand,alterations of cell volume have been shown to modify lysosomal pH,which is a determinant of LDL_b+i.LDL_b+i have been estimated fromheparin-releasable (binding) or heparin-insensitive (internalization)uptake of ¹²⁵I-labeled LDL. OSMewas modified by alterations of extracellular concentrations of ions,glucose, urea, or raffinose. When OSMe was altered by varying NaClconcentrations, LDL_b+i decreased (by 0.5 ± 0.1%/mM) with increasing OSMe andLDL_b+i increased (by 1.2 ± 0.1%/mM) with decreasing OSMe, an effect mainly due to alteredaffinity; the estimated dissociation constant amounted to 20.6, 48.6, and 131.6 µg/ml at 219, 293, and 435 mosM, respectively. A 25%increase of OSMe increased cytosolic (by 0.46 ± 0.03) and decreasedlysosomal (by 0.14 ± 0.02) pH. Conversely, a 25% decrease of OSMedecreased cytosolic (by 0.28 ± 0.02) and increased lysosomal (by0.17 ± 0.02) pH. Partial replacement of extracellularNa⁺ withK⁺ had little effect onLDL_b+i, although it swelledhepatocytes and increased lysosomal and cytosolic pH. Hypertonicglucose, urea, or raffinose did not exert similar effects despite ashrinking effect of hypertonic raffinose. Monensin, which completelydissipates lysosomal acidity, virtually abolishedLDL_b+i. In conclusion, theobservations reveal a significant effect of ionic strength onLDL_b+i. The effect is, however,not likely to be mediated by alterations of cell volume or alterationsof lysosomal pH.

     

Dual effect of nitric oxide on cytosolic Ca2+ concentration and insulin secretion in rat pancreatic beta-cells

Inisolated rat pancreatic -cells, the nitric oxide (NO) donor NOC-7 at1 µM reduced the amplitude of the oscillations of cytosolicCa²⁺ concentration ([Ca²⁺]_c)induced by 11.1 mM glucose, and at 10 µM terminated them. In thepresence of N^G-nitro-L-arginine(L-NNA), however, NOC-7 at 0.5 and 1 µM increased theamplitude of the [Ca²⁺]_c oscillations,although the NO donor at 10 µM still suppressed them. Aqueous NOsolution also had a dual effect on the[Ca²⁺]_c oscillations. The soluble guanylatecyclase inhibitor LY-83583 and the cGMP-dependent protein kinaseinhibitor KT5823 inhibited the stimulatory effect of NO, and8-bromo-cGMP increased the amplitude of the[Ca²⁺]_c oscillations. Patch-clamp analyses inthe perforated configuration showed that 8-bromo-cGMP inhibited wholecell ATP-sensitive K⁺ currents in the isolated ratpancreatic -cells, suggesting that the inhibition by cGMP ofATP-sensitive K⁺ channels is, at least in part, responsiblefor the stimulatory effect of NO on the[Ca²⁺]_c oscillations. In the presence ofL-NNA, the glucose-induced insulin secretion from isolatedislets was facilitated by 0.5 µM NOC-7, whereas it was suppressed by10 µM NOC-7. These results suggest that NO facilitatesglucose-induced [Ca²⁺]_c oscillations of-cells and insulin secretion at low concentrations, which effectsare mediated by cGMP, whereas NO inhibits them in a cGMP-independentmanner at high concentrations.

     

K+-Na+ Exchange and Selectivity in Barley Root Cells: Effect of Na+ on the Na+ Fluxes

Using the compartmental analysis the unidirectional Na⁺ fluxesin cortical cells of barley roots, the cytoplasmic and vacuolarNa⁺ contents Q_c and Q_v, and the trans-root Na⁺ transport R'have been studied as a function of the external Na⁺ concentration.Using the re-elution technique the effect of low K⁺ concentrationson the plasmalemma efflux _co of Na⁺ (K+-Na+ exchange) and onR' was investigated at different Na+ concentrations and correspondinglydifferent values of the cytoplasmic sodium content Qc. The relationof the K+-dependent Na+ efflux coK+-dep to Qc or to the cytoplasmicNa+ concentration obeyed Michaelis-Menten kinetics. This isconsistent with a linkage of co, K+-dep to K+ influx by a K⁺-Na⁺exchange system. The apparent Km corresponded to a cytoplasmicNa⁺ concentration of 28 mM at 0·2 mM K⁺ and about 0·2mM Na⁺ in the external solution. 0·2 mM K⁺ stimulatedthe plasma-lemma efflux of Na⁺ and inhibited Na⁺ transport selectivelyeven in the presence of 10 mM Na⁺ in the external medium showingthe high efficiency of the K⁺-Na⁺ exchange system. However,_co, K⁺-dep was inhibited at 10 mM Na¹ compared to lower Na¹concentrations suggesting some competition of Na¹ with K¹ atthe external site of the exchange system. The effect of theNa+ concentration on Na¹ influx _oc is discussed with respectto kinetic models of uuptake.     

Characteristics of hyperpolarization-activated cation currents in portal vein smooth muscle cells

Voltage-clamp studies offreshly isolated smooth muscle cells from rabbit portal veinrevealed the existence of a time-dependent cation current evoked bymembrane hyperpolarization (termed I_h). Both therate of activation and the amplitude of I_h wereenhanced by membrane hyperpolarization. Half-maximal activation ofI_h was about 105 mV with conventional wholecell and 80 mV when the perforated patch technique was used. Incurrent clamp, injection of hyperpolarizing current produced a markeddepolarizing "sag" followed by rebound depolarization. Activationof I_h was augmented by an increase in theextracellular K⁺ concentration and was blocked rapidly byexternally applied Cs⁺ (1-5 mM). The bradycardic agentZD-7288 (10 µM), a selective inhibitor of I_h,produced a characteristically slow inhibition of the portal veinI_h. The depolarizing sag recorded in current clamp was also abolished by application of 5 mM Cs⁺.Cs⁺ significantly decreased the frequency of spontaneouscontractions in both whole rat portal vein and rabbit portal veinsegments. Multiplex RT-PCR of rabbit portal vein myocytes using primers derived from existing genes for hyperpolarization-activated cation channels (HCN1-4) revealed the existence of cDNA clonescorresponding to HCN2, 3, and 4. The present study shows that portalvein myocytes contain genes shown to encode forhyperpolarization-activated channels and exhibit an endogenous currentwith characteristics similar to I_h in other celltypes. This conductance appears to determine, in part, the rhythmicityof this vessel.

     

Isolated mouse pancreatic beta-cells show cell-specific temporal response pattern

The length of the silent lag time beforeelevation of the cytosolic free Ca²⁺ concentration([Ca²⁺]_i) differs between individualpancreatic -cells. One important question is whether thesedifferences reflect a random phenomenon or whether the length of lagtime is inherent in the individual -cell. We compared the lag times,initial dips, and initial peak heights for[Ca²⁺]_i from two consecutive glucosestimulations (with either 10 or 20 mM glucose) in individualob/ob mouse -cells with the fura 2 technique in amicrofluorimetric system. There was a strong correlation between thelengths of the lag times in each -cell (10 mM glucose:r = 0.94, P < 0.001; 20 mM glucose:r = 0.96, P < 0.001) as well as between theinitial dips in [Ca²⁺]_i (10 mM glucose:r = 0.93, P < 0.001; 20 mM glucose:r = 0.79, P < 0.001) and between theinitial peak heights (10 mM glucose: r = 0.51, P < 0.01; 20 mM glucose: r = 0.77, P < 0.001). These data provide evidence that theresponse pattern, including both the length of the lag time and thedynamics of the subsequent [Ca²⁺]_i, isspecific for the individual -cell.

     

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl^– channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V_Post-RVD < V_baseline). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl^– channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd²⁺ (V_Post-RVD > 1.4*V_baseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K⁺-Cl^– cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd²⁺ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl^– channels vs. transporters to this process is 60–70% and 30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl^– channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport