Diffusion of Oxyleghaemoglobin

The diffusion of oxyleghaemoglobin, prepared from soybean rootnodules, was measured at 24°C in agar and agarose gels ofvarious strengths, or in 1% agarose containing 0-18% (w/v) bovineserum albumin, to simulate the protein content of the cytoplasmof root nodule cells. Values of D_p, the diffusion coefficient,were unaffected (D_p = 11·8 x 10^-11 m² s^-1; s.e.m. 0·3x 10^-11) until the protein concentration exceeded 6%, abovewhich D_p declined sharply. With 18% bovine serum albumin, theconcentration of total soluble protein calculated to be presentin the cytoplasm of infected cells, where most of the leghaemoglobinis located in vivo, D_p was 5·9 x 10^-11 m² s^-1. Theseresults are discussed in relation to leghaemoglobin-facilitateddelivery of O₂ to the respiring N₂-fixing bacteroids in rootnodule cells.Copyright 1993, 1999 Academic Press Bacteroids, diffusion, Glycine max, N₂ fixation, oxyleghaemoglobin, soybean, root nodules     

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.

     

Modulation of K+ channels by arachidonic acid in T84 cells. I.Inhibition of the Ca2+-dependent K+ channel

TheCl secretory response ofcolonic cells to Ca²⁺-mediatedagonists is transient despite a sustained elevation of intracellular Ca²⁺. We evaluated the effects ofsecond messengers proposed to limit Ca²⁺-mediatedCl secretion on thebasolateral membrane,Ca²⁺-dependentK⁺ channel(K_Ca) in colonic secretorycells, T84. Neither protein kinase C (PKC) nor inositoltetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affectedK_Ca in excised inside-out patches.In contrast, arachidonic acid (AA; 3 µM) potently inhibitedK_Ca, reducingNP_o, the productof number of channels and channel open probability, by 95%. Theapparent inhibition constant for this AA effect was 425 nM. AAinhibited K_Ca in the presence ofboth indomethacin and nordihydroguaiaretic acid, blockers of thecyclooxygenase and lipoxygenase pathways. In the presence of albumin,the effect of AA on K_Ca wasreversed. A similar effect of AA was observed onK_Ca during outside-out recording.We determined also the effect of thecis-unsaturated fatty acid linoleate,the trans-unsaturated fatty acidelaidate, and the saturated fatty acid myristate. At 3 µM, all ofthese fatty acids inhibited K_Ca,reducing NP_o by 72-86%. Finally, the effect of the cytosolic phospholipaseA₂ inhibitorarachidonyltrifluoromethyl ketone(AACOCF₃) on thecarbachol-induced short-circuit current(I_sc) responsewas determined. In the presence ofAACOCF₃, the peakcarbachol-inducedI_sc response wasincreased ~2.5-fold. Our results suggest that AA generation inducedby Ca²⁺-mediated agonists maycontribute to the dissociation observed between the rise inintracellular Ca²⁺ evoked by theseagonists and the associatedCl secretory response.

     

Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel

Extracellular ATP is known to trigger apoptosis of thymocytesand lymphocytes through a P2Z receptor at which ATP is a partial agonist, giving only 70% of the maximum response of3'-O-(4-benzoyl)benzoyl-adenosine 5'-triphosphate (BzATP), a full agonist. This cytolytic receptor and its associated ion channel areCa²⁺ (andBa²⁺) selective but also passmolecules up to the size of ethidium cation (314 Da).RT-PCR showed identity between lymphocyte P2Z and thehP2X₇ gene recently cloned fromhuman monocytes. When human leukemic B lymphocytes were incubated withATP and¹³³Ba²⁺,an immediate influx of isotope occurred. It was augmented by 45% whenATP was added 10 min before isotope. Time-resolved flow cytometry wasused to examine kinetics of ethidium uptake in cells incubated withBzATP or the partial agonists ATP, 2-methylthioadenosine 5'-triphosphate, or adenosine5'-O-(3-thiotriphosphate).Maximally effective concentrations of BzATP (50 µM) induced immediateuptake of ethidium at a rate linear with time. In contrast, a delay was observed (30 s) before ethidium uptake commenced after addition ofmaximally effective ATP concentrations (500 µM) at 37°C, and thedelay was longer at 24°C. ATP addition 2-10 min beforeethidium abolished the delay. The delay was longer with other partialagonists and inversely related to maximal flux produced by agonist. Adelay was also observed for submaximal BzATP concentrations (10-20µM). P2Z/P2X₇ inhibitors, KN-62and5-(N,N-hexamethylene)-amiloride, reduced the rate of agonist-induced ethidium uptake and lengthened thedelay. The results support a model in which agonists forP2Z/P2X₇ receptor mediate animmediate channel opening allowing passage of small inorganic cations,followed by a slow further permeability increase allowing passage oflarger permeant cations like ethidium. The rate of the second stepdepends on time and temperature and the efficacy and concentration ofagonist and is slowed by antagonists, suggesting it depends on thefraction of P2Z/P2X₇ channels held in the initial openstate.

     

Liver fatty acid-binding protein gene ablation inhibits branched-chain fatty acid metabolism in cultured primary hepatocytes

Whereas the role of liver fatty acid-binding protein (L-FABP) in the uptake, transport, mitochondrial oxidation, and esterification of normal straight-chain fatty acids has been studied extensively, almost nothing is known regarding the function of L-FABP in peroxisomal oxidation and metabolism of branched-chain fatty acids. Therefore, phytanic acid (most common dietary branched-chain fatty acid) was chosen to address these issues in cultured primary hepatocytes isolated from livers of L-FABP gene-ablated (-/-) and wild type (+/+) mice. These studies provided three new insights: First, L-FABP gene ablation reduced maximal, but not initial, uptake of phytanic acid 3.2-fold. Initial uptake of phytanic acid uptake was unaltered apparently due to concomitant 5.3-, 1.6-, and 1.4-fold up-regulation of plasma membrane fatty acid transporter/translocase proteins (glutamic-oxaloacetic transaminase, fatty acid transport protein, and fatty acid translocase, respectively). Second, L-FABP gene ablation inhibited phytanic acid peroxisomal oxidation and microsomal esterification. These effects were consistent with reduced cytoplasmic fatty acid transport as evidenced by multiphoton fluorescence photobleaching recovery, where L-FABP gene ablation reduced the cytoplasmic, but not membrane, diffusional component of NBD-stearic acid movement 2-fold. Third, lipid analysis of the L-FABP gene-ablated hepatocytes revealed an altered fatty acid phenotype. Free fatty acid and triglyceride levels were decreased 1.9- and 1.6-fold, respectively. In summary, results with cultured primary hepatocytes isolated from L-FABP (+/+) and L-FABP (-/-) mice demonstrated for the first time a physiological role of L-FABP in the uptake and metabolism of branched-chain fatty acids.     

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.

     

Effect of concentration on albumin diffusion in lung interstitium

The transport of macromolecules through the lung interstitiumdepends on both bulk transport of fluid and diffusion. In the presentstudy, we studied the diffusion of albumin. Isolated rabbit lungs wereinflated with silicon rubber via airways and blood vessels, and twochambers were bonded to the sides of a 0.5-cm-thick slab that encloseda vessel with an intersititial cuff. One chamber was filled with eitheralbumin solution (2 or 5 g/dl) containing tracer¹²⁵I-albumin or with tracer¹²⁵I-albumin alone; the other wasfilled with Ringer solution. Unbound ¹²⁵I was removed from the tracerby dialysis before use. The chamber with Ringer solution was placed inthe well of a NaI(Tl) scintillation detector. Diffusion oftracer through the interstitium was measured continuously for 60 h.Tracer mass (M) showed a time(t) delay followed by an increase toa steady-state flow(dM/dtconstant). Albumin diffusion coefficient(D) was given byL²/(6T),where T was the time intercept of thesteady-stateM-t line at zero M, andL was interstitial length.Interstitial cuff thickness-to-vessel radius ratio(Th₀/R)was estimated by using Fick's law for steady-state diffusion. BothD andTh₀/Rwere independent of albumin concentration.D averaged 6.6 × 10⁷cm²/s, similar to the freeD for albumin. Values ofTh₀/Raveraged 0.047 ± 0.024 (SD), near the values measuredhistologically. Thus pulmonary interstitial constituents offered norestriction to the diffusion of albumin.

     

Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia

To study and define the early time-dependent response (6 h) ofblocker-sensitive epithelial Na⁺channels (ENaCs) to stimulation ofNa⁺ transport by aldosterone, weused a new modified method of blocker-induced noise analysis todetermine the changes of single-channel current (i_Na) channel open probability(P_o), andchannel density(N_T) undertransient conditions of transport as measured by macroscopic short-circuit currents(I_sc). In threegroups of experiments in which spontaneous baseline rates of transportaveraged 1.06, 5.40, and 15.14 µA/cm², stimulation of transportoccurred due to increase of blocker-sensitive channels.N_T variedlinearly over a 70-fold range of transport (0.5-35µA/cm²). Relatively small andslow time-dependent but aldosterone-independent decreases ofP_o occurredduring control (10-20% over 2 h) and aldosterone experimentalperiods (10-30% over 6 h). When theP_o of control andaldosterone-treated tissues was examined over the 70-fold extendedrange of Na⁺ transport,P_o was observedto vary inversely withI_sc, falling from~0.5 to ~0.15 at the highest rates ofNa⁺ transport or ~25% per3-fold increase of transport. Because decreases ofP_o from anysource cannot explain stimulation of transport by aldosterone, it isconcluded that the early time-dependent stimulation ofNa⁺ transport in A6 epithelia isdue exclusively to increase of apical membraneN_T.

     

Comparison between the uptake of nitrous oxide and nitric oxide in the human nose

The absorption of nitrous oxide(N₂O) during unidirectional flowwas compared with the rate of uptake of nitric oxide (NO). At flowrates of 10, 20, and 60 ml/min from one nostril to the other, with thesoft palate closed, the N₂Oreached a steady-state rate of absorption in 5-15 min. The meansuperficial capillary blood flow (n = 5) calculated from solubility and the steady-state rate ofN₂O absorption ranged from 13.3 to15.9 ml/min. The relation between absorption ofN₂O in the nose and capillaryblood flow fits a ventilation-perfusion model used by others todescribe uptake of inert, soluble gases in the rat nose. By contrast,the rate of uptake of NO gas, which is chemically reactive, is25-31 times as great as predicted by just its blood-to-airpartition coefficient. Exogenous NO (16.9 parts/million) did not induce nasal vasodilation as measured with laser Doppler andN₂O absorption methods. Thedifference between the measured rate of uptake of NO and the rate ofuptake attributable to its partition coefficient in blood at the rateof blood flow calculated from N₂Ouptake is probably due to chemical reaction of NO in mucous secretions, nasal tissues, and capillary blood.

     

小鼠胚胎干细胞分化心肌细胞毒蕈碱受体的表达特征

为了探讨胚胎干细胞分化心肌细胞（ESCM）毒蕈碱受体的表达规律及β肾上腺素能系统对M2受体表达的影响,采用10^－4 mol/L维生素C体外诱导小鼠M13胚胎干细胞分化为心肌细胞,用RT-PCR检测到分化后的细胞表达心肌细胞特异性基因Nkx2.5和β肌球蛋白重链;用免疫荧光法检测到分化后的细胞表达心肌细胞特异性标志物α辅肌动蛋白.小鼠胚胎干细胞分化前表达M₁和M₂毒蕈碱受体,在分化过程中,M₁受体表达逐渐下降, M₂受体表达在第3 d显著下降,此后表达逐渐增加,在第14 d达到高峰;Western印迹结果显示,异丙肾上腺素明显抑制M₂受体的表达,选择性β₁肾上腺素受体拮抗剂CGP20712A明显上调其表达,而选择性β₂肾上腺素受体拮抗剂 ICI118551对其表达无影响.本实验表明,小鼠胚胎干细胞分化心肌细胞表达毒蕈碱受体, β肾上腺素能系统对M₂受体表达有调控作用.     

Effects of fatty acids on BK channels in GH(3) cells

Ca²⁺-activated K⁺ (BK) channels inGH₃ cells are activated by arachidonic acid (AA). Becausecytosolic phospholipase A₂ can produce other unsaturatedfree fatty acids (FFA), we examined the effects of FFA on BK channelsin excised patches. Control recordings were made at several holdingpotentials. The desired FFA was added to the bath solution, and thevoltage paradigm was repeated. AA increased the activity of BK channelsby 3.6 ± 1.6-fold. The cis FFA, palmitoleic, oleic,linoleic, linolenic, eicosapentaenoic, and the triple bond analog ofAA, eicosatetraynoic acid, all increased BK channel activity, whereasstearic (saturated) or the trans isomers elaidic,linolelaidic, and linolenelaidic had no effect. The cisunsaturated FFA shifted the open probability vs. voltage relationshipsto the left without a change in slope, suggesting no change in thesensitivity of the voltage sensor. Measurements of membrane fluidityshowed no correlation between the change of membrane fluidity and thechange in BK channel activation. In addition, AA effects on BK channelswere unaffected in the presence of N-acetylcysteine.Arachidonyl-CoA, a membrane impermeable analog of AA, activateschannels when applied to the cytosolic surface of excised patches,suggesting an effect of FFAs from the cytosolic surface of BK channels.Our data imply a direct interaction between cis FFA and theBK channel protein.

     

N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X(7) receptors

Activation of membrane P2X₇ receptors by extracellular ATP [or its analog 2',3'-O-(4-benzoylbenzoyl)-ATP] results in the opening within several milliseconds of an integral ion channel that is permeable to small cations. If the ATP application is maintained for several seconds, two further sequelae occur: there is a gradual increase in permeability to the larger cation N-methyl-D-glucamine and the cationic propidium dye quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide (YO-PRO-1) enters the cell. The similarity in the time course of these two events has led to the widespread view that N-methyl-D-glucamine and YO-PRO-1 enter through a common permeation pathway, the "dilating" P2X₇ receptor pore. Here we provide two independent lines of evidence against this view. We studied single human embryonic kidney cells expressing rat P2X₇ receptors with patch-clamp recordings of membrane current and with fluorescence measurements of YO-PRO-1 uptake. First, we found that maintained application of the ATP analog did not cause any increase in N-methyl-D-glucamine permeability when the extracellular solution contained its normal sodium concentration, although YO-PRO-1 uptake was readily observed. Second, we deleted a cysteine-rich 18-amino acid segment in the intracellular juxtamembrane region of the P2X₇ receptor. This mutated receptor showed normal YO-PRO-1 uptake but had no permeability to N-methyl-D-glucamine. Together, the clear differential effects of extracellular sodium ions or of mutation of the receptor strongly suggest that N-methyl-D-glucamine and YO-PRO-1 do not enter the cell by the same permeation pathway. ATP; cation channel; permeability; quinolinium, 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide     

Contraction-induced increase in Vmax of palmitate uptake and oxidation in perfused skeletal muscle

To evaluate the effects of contractions on thekinetics of uptake and oxidation of palmitate in a physiological musclepreparation, rat hindquarters were perfused with glucose (6 mmol/l),albumin-bound [1-¹⁴C]palmitate, andvarying amounts of albumin-bound palmitate (200-2,200 µmol/l) atrest and during muscle contractions. When plotted against the unboundpalmitate concentration, palmitate uptake and oxidation displayedsimple Michaelis-Menten kinetics with estimated maximal velocity(V_max)and Michaelis-Menten constant(K_m) values of42.8 ± 3.8 (SE)nmol · min¹ · g¹and 13.4 ± 3.4 nmol/l for palmitate uptake and 3.8 ± 0.4 nmol · min¹ · g¹and 8.1 ± 2.9 nmol/l for palmitate oxidation, respectively, at rest.Whereas muscle contractions increased theV_maxfor both palmitate uptake and oxidation to 91.6 ± 10.1 and 16.5 ± 2.3 nmol · min¹ · g¹,respectively, theK_m remainedunchanged.V_maxand K_m estimates obtained from Hanes-Woolf plots (substrate concentration/velocity vs.substrate concentration) were not significantly different. In theresting perfused hindquarter, an increase in palmitate delivery from31.9 ± 0.9 to 48.7 ± 1.2 µmol · g¹ · h¹by increasing perfusate flow was associated with a decrease in thefractional uptake of palmitate so that the rates of uptake andoxidation of palmitate remained unchanged. It is concluded that therates of uptake and oxidation of long-chain fatty acids (LCFA) saturatewith an increase in the concentration of unbound LCFA in perfusedskeletal muscle and that muscle contractions, but not an increase inplasma flow, increase theV_maxfor LCFA uptake and oxidation. The data are consistent with the notion that uptake of LCFA in muscle may be mediated in part by a transport system.

     

Induction of Light Dependent Pyruvate Transport into Chloroplasts of Mesembryanthemum crystallinum by Salt Stress

Mode of photosynthesis in Mesembryanthemum crystallinum changesfrom C₃ to Crassulacean acid metabolism (CAM) when the plantswere stressed with high salinity. [¹⁴C]Pyruvate uptake for 30s into intact chloroplasts isolated from leaves of the CAM modeof M. crystallinum was enhanced more than 5-fold in the lightcompared with that in the dark. The stromal concentration ofpyruvate in the light reached to more than 2.5 times of themedium. In contrast, little or no pyruvate uptake occurred inchloroplasts from C₃ leaves in either light or dark condition.The initial uptake rate (10 s incubation at 4°C) into theCAM chloroplasts in the light was about 3-fold higher than therate in the dark. K_m and V_max of the initial uptake in the lightwere 0.54 mM and 8.5 µmol (mg Chl)^–1 h^–1 respectively.These suggest that pyruvate was actively incorporated into theCAM chloroplasts against its concentration gradient across theenvelope in the light. When hydroponically grown M. crystallinumwere stressed by 350 mM NaCl, the capacity of chloroplasts forpyruvate uptake was induced in 6 d corresponding to the inductionof the activities of PEP-carboxylase and NAD(P)⁺-malic enzymesin response to salt stress. (Received October 12, 1995; Accepted January 19, 1996)     

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.

     

Phosphate transport by the human renal cotransporter NaPi-3 expressed in HEK-293 cells

The human renal Na-PO4cotransporter gene NaPi-3 was expressed in human embryonic kidneyHEK-293 cells, and the transport characteristics were measured in cellstransfected with a vector containing NaPi-3 or with the vector alone(sham transfected). The initial rate of32PO4influx had saturation kinetics for external Na andPO4 with K Na1/2 of 128 mM(PO4 = 0.1 mM) andK PO41/2of 0.084 mM (extracellular Na = 143 mM) in sham- and NaPi-3-transfectedcells expressing the transporter. Transfection had no effect on theNa-independent 32PO4influx, but transfection increased Na-dependent32PO4influxes 2.5- to 5-fold. Of the alkali cations, only Na significantly supported PO4 influx. Arsenateinhibited flux with an inhibition constant of 0.4 mM. The phosphatetransport in sham- and NaPi-3-transfected cells has nearly the sametemperature dependence in the absence and presence of extracellularNa. The Na-dependent phosphate flux decreased with pH insham-transfected cells but was pH independent in transfected cells. TheNa-dependent32PO4influx was inhibited byp-chloromercuriphenylsulfonate,phosphonoformate, phloretin, vanadate, and5-(N-methyl-N-isobutyl)-amiloridebut not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known behavior ofNaPi-3 homologues in the renal tubule of other species and, thus,demonstrate the fidelity of this transfection system for the study ofthis protein. Commensurate with the increased functional expression,there was an increase in the amount of NaPi-3 protein by Westernanalysis.

     

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.

     

Expression of smooth muscle myosin light chain 17 and unloaded shortening in single smooth muscle cells

These experiments were performed totest the hypotheses that myosin light chain 17 (MLC₁₇) aand b isoform expression varies between individual vascular smoothmuscle (SM) cells and that their expression correlates with cellunloaded shortening velocity. Single SM cells isolated from rabbitaorta and carotid arteries were used to measure unloaded shorteningvelocity and subsequently were analyzed via RT-PCR forMLC₁₇ a and b mRNA ratio. The MLC_17b/a mRNA andprotein ratios from adjacent tissue sections correlate very well(R² = 0.68), allowing use of the mRNA ratio topredict the protein ratio. The rabbit MLC₁₇ isoform proteinsequence was found to be similar to, but unique from, the swine, mouse,and chicken sequences. Isolated single SM cells from the aorta andcarotid have resting lengths of 70-280 µm and shorten to33-88 µm after contraction. Isolated cell maximum unloadedshortening velocity is highly variable (0.5-7.5 µm/s) butbecomes more uniform when normalized to initial cell length(0.01-0.05 cell lengths/s). Carotid cells activated in thepresence of okadaic acid (1 µm) have mean maximal unloaded shorteningvelocities not significantly different from carotid cells activatedwithout okadaic acid (0.016 vs. 0.019 cell lengths/s). Resting celllength before activation is significantly correlated with final celllength after unloaded shortening. Neither initial cell length, finalcell length, total cell length change, nor maximum unloaded shorteningvelocity (absolute or normalized) was significantly correlated withsingle-cell MLC_17b/a mRNA ratio. These studies wereperformed in isolated single SM cells where unloaded shorteningvelocity and MLC_17b/a mRNA ratios were measured in the samecell. In this preparation, the three-dimensional organization andmilieu of the cell is kept intact, but without the intercellularheterogeneity concerns of multicellular preparations. These resultssuggest the MLC_17b/a ratio is variable between individual SM cells from the same tissue, but it is not a determinant of unloadedshortening velocity in single SM cells.

     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.

     

Influence of increased mechanical loading by hypergravity on the microtubule cytoskeleton and prostaglandin E2 release in primary osteoblasts

Cells respond to a wide range of mechanical stimuli such as fluid shear and strain, although the contribution of gravity to cell structure and function is not understood. We hypothesized that bone-forming osteoblasts are sensitive to increased mechanical loading by hypergravity. A centrifuge suitable for cell culture was developed and validated, and then primary cultures of fetal rat calvarial osteoblasts at various stages of differentiation were mechanically loaded using hypergravity. We measured microtubule network morphology as well as release of the paracrine factor prostaglandin E₂ (PGE₂). In immature osteoblasts, a stimulus of 10x gravity (10 g) for 3 h increased PGE₂ 2.5-fold and decreased microtubule network height 1.12-fold without affecting cell viability. Hypergravity (3 h) caused dose-dependent (5–50 g) increases in PGE₂ (5.3-fold at 50 g) and decreases (1.26-fold at 50 g) in microtubule network height. PGE₂ release depended on duration but not orientation of the hypergravity load. As osteoblasts differentiated, sensitivity to hypergravity declined. We conclude that primary osteoblasts demonstrate dose- and duration-dependent sensitivity to gravitational loading, which appears to be blunted in mature osteoblasts. mechanotransduction; differentiation; bone