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; )     

Effect of NaCI Salinity on Flows and Partitioning of C, N, and Mineral Ions in Whole Plants of White Lupin, Lupinus albusL.

Plants of Lupinus albus L., cv. Ultra, were grown hydroponicallywith NO₃^–-nutrition for 51 d under control (0.05 mol m^–3Na⁺ and 10 mol m^–3 Cl^–) and saline (40 mol m^–3NaCI) conditions. Plants were harvested 41 and 51 d after germinationand analysed for content and net increment of C, N and the mineralcations K⁺, Na⁺, Mg²⁺, and Ca²⁺ and the anions Cl^–, NOJ,malate, phosphate, and SO₄^2–. Roots, stem interaodes,petioles and leaflets were analysed separately. During the studyperiod net photosynthesis, respiratory losses of CO₂ from shootand root and the composition of the spontaneously bleeding phloemsap and the root pressure xylem exudate were also determined.Using molar ratios of C over N in the transport fluids, incrementsof C and N, and photosynthetic gains as well as respiratorylosses of C, the net flows of C and N in the xylem and phloemwere then calculated as in earlier studies (Pate, Layzell andMcNeill, 1979a). Knowing the carbon flows, the ratios of ionto carbon in the phloem sap, and ion increments in individualorgans, net flows of K⁺, Na⁺, and Cl^– over the study periodwere also calculated. Salt stress led to a general decrease of all partial componentsof C and N partitioning indicating that inhibitions were notdue to specific effects of NaCI salinity on photosynthesis oron NO₃ uptake. However, there were differences between variouslyaged organs, and net phloem export of nitrogenous compoundsfrom ageing leaves was substantially enhanced under saline conditions.In addition, NO₃^–reduction in the roots was specificallyinhibited. Uptake and xylem transport of K⁺ was more severelyinhibited than photosynthetic carbon gain or NO₃^– uptakeby the root. K⁺ transport in the phloem was even more severelyrestricted under saline conditions. Na⁺ and Cl^– flowsand uptake, on the other hand, were substantially increasedin the presence of salt and, in particular, there were thenmassive flows of Na^– in the phloem. The results are discussedin relation to the causes of salt sensitivity of Lupinus albus.The data suggest that both a restriction of K⁺ supply and astrongly increased phloem translocation of Na⁺ contribute tothe adverse effects of salt in this species. Restriction ofK⁺ supply occurs by diminished K⁺ uptake and even more by reducedK⁺ cycling within the plant. Key words: Lupinus albus, salt stress, phloem transport, xylem transport, partitioning, carbon, nitrogen, K⁺, Na⁺, CI^–     

Ion Composition of the Chara Internode

Ion compositions of the cytoplasm and the vacuole of Chara australiswere analyzed according to Kishimoto and Tazawa (1964) and Kiyosawa(1979a). The ions in the cytoplasm and the vacuole analyzedwere K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, NO₃^– and H₂PO₄^–.Assuming that the volume of the cytoplasm V_p is 10% of thatof the whole cell V, the concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺,Cl^–, NO₃^– and H₂PO₄^– in the cytoplasm averaged70, 15, 13, 4.6, 31, 2.2 and 16 mM, respectively. If the volumeof the cytoplasm was assumed to be 5% of that of the whole cell,their averaged concentrations were 139, 31, 25, 9.2, 62, 4.4and 33 mM, respectively. The averaged ion compositions of thecell sap were K⁺, 111; Na⁺, 47; Ca²⁺, 4.4; Mg²⁺, 8.9; Cl^–,91; NO^–₃, 3.3 and H₂PO₄^–, 6.0 mM. These values,taking the concentrations and the charges of the protein (Kiyosawa1979b) and amino acids (Sakano and Tazawa 1984) into accountand assuming the presence of some uni- or oligovalent anionsand/or small nonelectrolyte molecules, could explain fairlywell both the electroneutrality and the osmotic pressure ofthe cell, except when V_p/V = 5%. (Received May 18, 1987; Accepted September 29, 1987)     

Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

Decoding of fast cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial [Ca²⁺] ([Ca²⁺]_m) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial [Ca²⁺] ([Ca²⁺]_em) to >0.5 µM resulted in a [Ca²⁺]_em-dependent increase in the rate of mitochondrial Ca²⁺ accumulation ([Ca²⁺]_em resulting in half-maximal rate of Ca²⁺ accumulation = 4.4 µM) via Ca²⁺ uniporter. Ca²⁺ uptake was sensitive to the Ca²⁺ uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of [Ca²⁺]_m increase and recovery were dependent on the extramitochondrial [Na⁺] ([Na⁺]_em) due to Ca²⁺ extrusion via mitochondrial Na⁺/Ca²⁺ exchanger. The maximal rate of Ca²⁺ extrusion was observed with [Na⁺]_em in the range of 20–40 mM. Rapid switching (0.25–1 Hz) of [Ca²⁺]_em between 0 and 100 µM simulated rapid beat-to-beat changes in [Ca²⁺]_i (with [Ca²⁺]_i transient duration of 100–500 ms). No [Ca²⁺]_m oscillations were observed, either under conditions of maximal rate of Ca²⁺ uptake (100 µM [Ca²⁺]_em, 0 [Na⁺]_em) or with maximal rate of Ca²⁺ removal (0 [Ca²⁺]_em, 40 mM [Na⁺]_em). The slow frequency-dependent increase of [Ca²⁺]_m argues against a rapid transmission of Ca²⁺ signals between cytosol and mitochondria on a beat-to-beat basis in the heart. [Ca²⁺]_m changes elicited by continuous or pulsatile exposure to elevated [Ca²⁺]_em showed no difference in mitochondrial Ca²⁺ uptake. Thus in cardiac myocytes fast [Ca²⁺]_i transients are integrated by mitochondrial Ca²⁺ transport systems, resulting in a frequency-dependent net mitochondrial Ca²⁺ accumulation. mitochondrial Ca²⁺; excitation-contraction coupling; cardiomyocytes     

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.

     

Diurnal K+ and Anion Transport in Phaseolus Pulvinus

Diurnal movement of Phaseolus leaf is caused by deformationof the laminar pulvinus located at the joint of the leaf bladeand the petiole. The plants were cultured in solutions withvarious ion compositions, and changes of K⁺, Na⁺, Ca²⁺, Mg²⁺,Cl^–, NO₃– and P₁ concentrations both in the upperand lower parts of the laminar pulvinus were measured. Culturein 10 mM KCl solution caused an increase in K⁺ and Cl^–concentrations both in the upper and lower parts without anysignificant change in the concentration of NO₃^–; culturein 10 mM KNO₃ solution caused an increase in K⁺ and NO₃^–concentration without any significant change in the concentrationof Cl^–; and culture in 10 mM KH₂PO₄ solution caused anincrease in K⁺ and P₁ concentrations without any significantchange in the concentrations of NO₃- and Cl^–. K⁺ moved from the upper to lower parts or from the lower toupper parts diurnally in all plants cultured in any solutionmentioned above. The main inorganic anion that accompanied thisK⁺ movement was Cl^– in KCl solution, and NO₃^– inKNO₃ solution. When the seedlings were cultured in distilledwater or in KH₂PO₄ solution, neither Cl^– NO₃^– norP₁ accompanied this K⁺ movement. In these cases, mainly H⁺ and/ororganic anions are supposed to move in exchange for and/or incombination with K⁺ movement. (Received November 8, 1982; Accepted June 13, 1983)     

Osmotic and Ionic Regulation in Chara L-cell Fragments

Ion absorption from rather complicated artificial pond water(APW) by cell fragments having a lower osmotic pressure thanthe intact internodal cell (L-cell fragments) was studied. L-cellfragments were prepared by taking advantage of trans cellularosmosis and ligating the cell with thread. The results wereas follows: (1) L-cell fragments absorbed more K⁺ than Na⁺ fromaKCL + NaCl mixture in the presence of Ca²⁺, Mg²⁺ and SO₂₄ inthe light; (2) the influx of KCI was larger than that of KNO₃;(3) the amount of positive charge carried by K⁺, Na⁺ and Mg²⁺across the cell membrane balanced well with the amount of negativecharge carried by Cl^– in Cl^–-containing and NO₃^–-free APW; (4) no conclusion could be made as to whether ornot the rule of electro neutrality held for the K⁺, Na⁺, Ca²⁺and NO₃^– fluxes across the cell membrane, because dilutedKNO₃ is unstable; (5) L-cell fragments in KCl-containing APWsurvived longer than those in KNO₃-containing and Cl^–-free APW; (6) after incubation in KNO₃-containing and Cl^–-freeAPW, L-cell fragments absorbed a great amount of KCI immediatelyafter being transferred to KCl-containing and NO₃^– -freeAPW; and (7) lowering the turgor pressure of the intact cellby raising the external osmotic pressure did not induce ionflux into the cell. Thus, we concluded that the L-cell fragmentsabsorbed ions from the external solution not because of theirlower turgor pressure, but because of the diluted ion concentrationof the cytoplasm and the vacuole. The electroneutrality ruleheld, at least, for K⁺, Na⁺, Mg²⁺ and Cl^– influxes acrossthe cell membrane inthe KCl-containing and NO₃^–-free APW.These results were analyzed on the basis of an extended poremodel which presumed the existence of ATP-dependent processesin the membrane, and suggested that K⁺, Na⁺ and Mg₂₊ inflowsinto an L-cell fragment are likely to be induced by active Cl^–inflow. (Received May 18, 1987; Accepted September 29, 1987)     

Unequal distribution of potassium and anions within the Phaseolus pulvinus during circadian leaf movement

Ion and saccharide concentrations in the upper and lower partsof the laminar pulvinus of the primary leaf of Phaseolus vulgariswere measured in relation to the circadian movement. Concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^–, organic acid,NO₃^–, H₂PO₄^–, fructose and fructose-yielding saccharidesin the pulvinus were 75–120, 0.3–0.7, 5–8,6–12, 40–60, 60–73, 19–35, 2–9and 1–5 mM, respectively, and the osmotic pressure ofthe pulvinus was considered to be due to these ions. The cell volume in the expanding part was larger than that inthe contracting part. The change of the cell volume alteredthe molar concentration in the cell sap and therefore the amountof solutes actually transported from the upper to the lowerpart and vice versa was estimated from the concentration expressedin moles per gram of dry weight. Results showed that K⁺, Cl^–, organic acid (or H⁺) andNO₃^– moved from the upper to lower parts or vice versain the pulvinus in relation to its deformation, keeping theelectroneutrality among those ions, whereas C_a2⁺ and Mg²⁺ didnot move. The difference in the K⁺ concentration between theupper and lower parts when the leaf was up or down amountedto 30% of the whole osmotic pressure. This lead to the conclusionthat the endogenous clock-controlled unequal distribution ofK⁺, Cl^–, organic acid (or H⁺) and NO₃^– in the pulvinuscould be the force for the circadian leaf movement. (Received August 7, 1979; )     

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells

Cl^– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is Cl^– dependent. After incubating the cells at different extracellular Cl^– concentration ([Cl^–]_e) for 40 min, the ANG II-induced Ca²⁺ transients at 120 meq/l Cl^– were more than twice those at either 80 or 20 meq/l Cl^–. Replacing Cl^– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca²⁺, ANG II-induced as well as platelet-derived growth factor-induced Ca²⁺ release exhibited Cl^– dependency. The difference of Ca²⁺ release with high vs. low [Cl^–]_e was not affected by acutely altering [Cl^–]_e 1 min before administration of ANG II when [Cl^–]_i was yet to be equilibrated with [Cl^–]_e. Pretreatment of a Cl^– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca²⁺ release and entry at 20 meq/l Cl^– but did not alter those at 120 meq/l Cl^–. However, after equilibration, a reduced [Cl^–]_e did not affect thapsigargin-induced Ca²⁺ release, suggesting that Cl^– may not affect the size of intracellular Ca²⁺ stores. Nevertheless, at high [Cl^–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] induced by ANG II was approximately sixfold that at low [Cl^–]. Thus the Cl^–-dependent effects of ANG II on Ca²⁺ transients may be mediated, at least in part, by a Cl^–-dependent Ins(1,4,5)P₃ accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca²⁺ release     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

Dependence of Plasmalemma Conductance and Potential on Intracellular Free Ca2+ in Tonoplast-Removed Cells of a Brackish Water Characeae Lamprothamnium

In response to hypotonic treatment internodal cells of the brackishwater Characeae Lamprothamnium regulate turgor pressure by releasingK⁺ and Cl^–, accompanying membrane depolarization and atransient increase in membrane electrical conductance (Okazakiet al. 1984b). The hypothesis that a transient increase in cytoplasmicfree Ca²⁺ concentration ([Ca²⁺]_c) caused by hypotonic treatmenttriggers release of K⁺ and Cl^– from the cell (Okazakiand Tazawa 1986a, b, c) was tested using tonoplast-removed cells.These cells did not regulate turgor pressure. The plasmalemmaconductance remained almost constant for a change in the intracellularfree Ca²⁺ concentration ([Ca²⁺],) from 10^–6 to 10^–2mol?m^–3. The results suggest that some cytoplasmic Ca²⁺-sensitizingsoluble components, which work as mediators to activate K⁺ and/orCl^– channels in the plasmalemma and/or the tonoplast,were lost after desintegration of the tonoplast. The plasmalemmapotential was depolarized under high [Ca²⁺]_i. However, no membranedepolarization was observed upon hypotonic treatment. Sincemembrane depolarization has been suggsted to occur under normal[Ca²⁺]_c in intact cells (Okazaki and Tazawa 1986a, b), its absencesuggests that some cytoplasmic factors, which induce the membranedepolarization in a Ca²⁺-independent manner, are lost in tonoplast-removedcells. ¹ Present address: Department of Biology, Osaka Medical College,Sawaragi-cho 2-41, Takatsuki, Osaka 569, Japan. (Received October 22, 1986; Accepted March 31, 1987)     

Palytoxin-induced cell death cascade in bovine aortic endothelial cells

The plasmalemmal Na⁺-K⁺-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca²⁺. However, the ability of PTX to directly increase cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) via Na⁺ pump channels and to initiate Ca²⁺ overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca²⁺]_i and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3–100 nM) produced a graded increase in [Ca²⁺]_i that was dependent on extracellular Ca²⁺. The increase in [Ca²⁺]_i initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC₅₀ < 1 µM. The elevation in [Ca²⁺]_i could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca²⁺]_i occurs via the Na⁺ pump. Subsequent to the rise in [Ca²⁺]_i, PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca²⁺ or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca²⁺ overload. necrosis; vital dyes; membrane blebs; time-lapse video microscopy; fura-2     

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.

     

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.

     

Protective role of extracellular chloride in fatigue of isolated mammalian skeletal muscle

A possible role of extracellular Cl^– concentration ([Cl^–]_o) in fatigue was investigated in isolated skeletal muscles of the mouse. When [Cl^–]_o was lowered from 128 to 10 mM, peak tetanic force was unchanged, fade was exacerbated (wire stimulation electrodes), and a hump appeared during tetanic relaxation in both nonfatigued slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. Low [Cl^–]_o increased the rate of fatigue 1) with prolonged, continuous tetanic stimulation in soleus, 2) with repeated intermittent tetanic stimulation in soleus or EDL, and 3) to a greater extent with repeated tetanic stimulation when wire stimulation electrodes were used rather than plate stimulation electrodes in soleus. In nonfatigued soleus muscles, application of 9 mM K⁺ with low [Cl^–]_o caused more rapid and greater tetanic force depression, along with greater depolarization, than was evident at normal [Cl^–]_o. These effects of raised [K⁺]_o and low [Cl^–]_o were synergistic. From these data, we suggest that normal [Cl^–]_o provides protection against fatigue involving high-intensity contractions in both fast- and slow-twitch mammalian muscle. This phenomenon possibly involves attenuation of the depolarization caused by stimulation- or exercise-induced run-down of the transsarcolemmal K⁺ gradient. potassium; skeletal muscle contraction; membrane potential; myotonia     

Ionic composition and pH of the vacuolar sap in marine dinoflagellate Noctiluca

Ionic composition of the vacuolar sap of Noctiluca miliariswas as follows: [Na⁺] = 487.3 mM, [K⁺]=24.1 mM, [Ca²⁺]=6.6 mM,[Mg²⁺]=2.8 mM, [Cl^–]=500mM, [NH₄⁺]=15–25 mM, and[SO₄^2–]=undetectable. To measure the vacuolar pH of singleliving cells, a pH-sensitive glass microelectrode was used.The vacuolar pH value was 3.50 ±0.18. When the cellswere transferred from normal sea water into osmotically adjusted50% sea water for one day, the vacuolar ion concentrations remainedalmost constant. Upon immersing the cells in osmotically unadjustedsea water of various concentrations for one day, the observedincrements or decrements of the vacuolar ion concentrationscould be accounted for largely by the migration of water outof or into the cells. The intrinsic ionic composition of thevacuole seems to be constant against changes in ion concentrationsof the bathing medium. (Received October 20, 1975; )     

Na+, K+ and Cl- in Xylem Sap Flowing to Shoots of NaCl-Treated Barley

Munns, R. 1985. Na⁺, K⁺ and Cl^– in xylem sap flowing toshoots of NaCl-treated barley.—J. exp. Bot. 36: 1032–1042. Na⁺, Cl^– and K⁺ concentrations were measured in xylemsap obtained by applying pressure to the roots of decapitatedbarley plants grown at external [NaCl] of 0, 25, 50, 100, 150and 200 mol m^–3. For any given NaCl treatment, ion concentrationsin the xylem sap were hyperbolically related to the flux ofwater. Ion concentrations in sap collected at very low volumefluxes (without applied pressure) were 5–10 times higherthan in sap collected at moderate fluxes (under pressure). Fora given moderate volume flux, Na+ concentration in the xylemsap, [Na⁺]_x, was only 4.0 mol m^–3 at external [NaCl] of25–150 mol m^–3, and increased to 7.0 mol m^–3at 200 mol m^–3. [Cl-]x showed a similar pattern. Thisshows there would be little difference in the rate of uptaketo the shoot of plants at 25–150 mol m^–3 externalNaCl and indicates little change even at 200 mol m^-3 NaCl becausetranspiration rates would be much lower. Thus the reduced growthof the shoot of plants at high NaCl concentrations is not dueto higher uptake rates of Na⁺ or Cl^–. The fluxes of Na⁺, Cl^– and K^– increased non-linearlywith increasing volume flux indicating little movement of saltin the apoplast. The flux of K⁺ increased even when [K⁺]_x wasgreater than external [K⁺], indicating that membrane transportprocesses modify the K⁺ concentration in the transpiration streamas it flows through the root system. Key words: -Xylem sap, Na⁺, K⁺, Cl^– fluxes, salinity, barley     

Distinct roles of PMCA isoforms in Ca2+ homeostasis of bladder smooth muscle: evidence from PMCA gene-ablated mice

We previously showed that plasma membrane Ca²⁺-ATPase (PMCA) activity accounted for 25–30% of relaxation in bladder smooth muscle (8). Among the four PMCA isoforms only PMCA1 and PMCA4 are expressed in smooth muscle. To address the role of these isoforms, we measured cytosolic Ca²⁺ ([Ca²⁺]_i) using fura-PE3 and simultaneously measured contractility in bladder smooth muscle from wild-type (WT), Pmca1^+/–, Pmca4^+/–, Pmca4^–/–, and Pmca1^+/–Pmca4^–/– mice. There were no differences in basal [Ca²⁺]_i values between bladder preparations. KCl (80 mM) elicited both larger forces (150–190%) and increases in [Ca²⁺]_i (130–180%) in smooth muscle from Pmca1^+/– and Pmca1^+/–Pmca4^–/– bladders than those in WT or Pmca4^–/–. The responses to carbachol (CCh: 10 µM) were also greater in Pmca1^+/– (120–150%) than in WT bladders. In contrast, the responses in Pmca4^–/– and Pmca1^+/–Pmca4^–/– bladders to CCh were significantly smaller (40–50%) than WT. The rise in half-times of force and [Ca²⁺]_i increases in response to KCl and CCh, and the concomitant half-times of their decrease upon washout of agonist were prolonged in Pmca4^–/– (130–190%) and Pmca1^+/–Pmca4^–/– (120–250%) bladders, but not in Pmca1^+/– bladders with respect to WT. Our evidence indicates distinct isoform functions with the PMCA1 isoform involved in overall Ca²⁺ clearance, while PMCA4 is essential for the [Ca²⁺]_i increase and contractile response to the CCh receptor-mediated signal transduction pathway. PMCA; bladder smooth muscle; gene-altered mice     

The Effects of Salinity upon Ion Content and Ion Transport of the Marine RedAlga Porphyra purpurea(Roth) C.Ag.

Ion contents and concentrations (K⁺, Na⁺, Cl^–, Ca²⁺, Mg²⁺,SO^2–₄, NO^–₃, HPOJ^2–₄, amino and organic acids)of P. purpurea have been studied in relation to salinity variation.Cells were shown to accumulate large amounts of K⁺ and Cl^–against their respective gradients of electrochemical potentialin all dilute and concentrated seawater media. Active influxof SOJ^2–₄, NO^–₃, and HPOJ^2–₄ is also suggested,while Na⁺ is actively excluded from cells under hyposaline andhypersaline conditions. The relative proportions of individualcomponents of the internal osmotic potential were found to changeaccording to the external salt concentration. KCL forms themajor fraction of _j} in concentrated seawater media while K⁺-aminoacids form the major fraction in dilute seawaters. Other intracellularsolutes comprise less than 15% oft_j, in all media. Unidirectional fluxes of K⁺ and Cl^– were studied by radioisotopicmeans. Fluxes of K⁺ and Cl^– are reduced in hyposalinemedia, as is absolute KCL content per cell. Intracelhilar KCLcontent was also found to be markedly dependent upon externalK⁺ concentration, rather than water potential. Changes in KC1levels induced by salinity variation occur over a 6 h period.     

The Ionic Relations of Acetabularia mediterranea

The concentrations of K⁺, Na⁺, and Cl^– in the cytoplasmand the vacuole of Acetabularia mediterranea have been measured,as have the vacuolar concentrations of SO₄^–– andoxalate. The electrical potential difference between externalsolution, and vacuole and cytoplasm has been measured. The resultsindicate that Cl^– and SO₄^–– are probably transportedactively into the cell, and that active transport of Na⁺ isoutwards. The results for K⁺ are equivocal. The fluxes of K⁺,Na⁺, Cl^–, and S0₄^–– into the cell and theeffluxes of Na⁺ and Cl^– have been determined. The Cl^–fluxes are extremely large. In all cases the plasmalemma isthe rate-limiting membrane for ion movement. A technique isdescribed for the preparation of large, completely viable cellfragments containing only cytoplasm, with no vacuole.