Volume Reabsorption, Transepithelial Potential Differences, and Ionic Permeability Properties in Mammalian Superficial Proximal Straight Tubules

This paper describes experiments designed to evaluate Na⁺ and Cl^- transport in isolated proximal straight tubules from rabbit kidneys. When the perfusing solution was Krebs-Ringer buffer with 25 mM HCO₃^- (KRB) and the bath contained KRB plus 6% albumin, net volume reabsorption (J_v, nl min^-1 mm^-1 was -0.46 ± 0.03 (SEM); V_e, the spontaneous transepithelial potential difference, was -1.13 ± 0.05 mV, lumen negative. Both J_v, and V_e, were reduced to zero at 21°C or with 10^-4 M ouabain, but J_v, was not HCO₃^- dependent. Net Na⁺ reabsorption, measured as the difference between ²²Na⁺ fluxes, lumen to bath and bath to lumen, accounted quantitatively for volume reabsorption, assuming the latter to be an isotonic process, and was in agreement with the difference between lumen to bath ²²Na⁺ fluxes during volume reabsorption and at zero volume flow. The observed flux ratio for Na⁺ was 1.46, and that predicted for a passive process was 0.99; thus, Na⁺ reabsorption was rationalized in terms of an active transport process. The Cl^- concentration of tubular fluid rose from 113.6 to 132.3 mM during volume reabsorption. Since V_e, rose to +0.82 mV when tubules were perfused with 138.6 mM Cl^- solutions, V_e may become positive when tubular fluid Cl^- concentrations rise during volume reabsorption. The permeability coefficients P_Na and P_Cl computed from tracer fluxes were, respectively, 0.23 x 10^-4 and 0.73 x 10^-4 cm s^-1. A P_Na/P_Cl ratio of 0.3 described NaCl dilution potentials at zero volume flow. The magnitudes of the potentials were the same for a given NaCl gradient in either direction and P_Na/P_Cl was constant in the range 32–139 mM NaCl. We infer that the route of passive ion permeation was through symmetrical extracellular interfaces, presumably tight junctions, characterized by neutral polar sites in which electroneutrality is maintained by mobile counterions.     

The relationship between sodium,potassium, and chloride in amphibian muscle

The Na,⁺ Cl^-, and K⁺ content of toad plasma and the sartorius muscle has been determined. Although the Na⁺ and Cl^- level of the muscles in the living animal varied greatly (0 to 38.0 m.eq. per kg., and 0 to 31.8 m.eq. per kg. respectively) the K⁺ level was subject to a smaller variation (76.5 to 136 m.eq. per kg.). There was a direct relationship between Na⁺ and Cl^-, which was independent of the K⁺ level. There is a closely related gain of Na⁺ and Cl^- when muscle is soaked in normal Ringer. These gains are not related to the K⁺ loss, frequently found on soaking. The relationship between the three ions was studied in a large series of 124 muscles in normal Ringer. As found in vivo, there was a correlation between Na⁺ and Cl.^- This correlation was independent of K⁺ content, except when this was abnormally low. Alteration of the external NaCl level produced concomitant changes in the internal levels of these ions. Alteration of the external KCl level produced an increase in internal Cl^- similar to that found with high NaCl solutions, but the amount of K⁺ entering the cell was approximately one-third of the external increase. Removal of K⁺ from the external solution did not result in a loss of K⁺ from the cell, although there was an adequate amount of Cl^- present to accompany it. The results cannot be reconciled with either a Donnan concept for the accumulation of K⁺, or a linked carrier system. A theory is proposed to account for the ionic differentiation within the cell. The K⁺ is assumed to be adsorbed onto an ordered intracellular phase. The normal metabolic functioning of the cell is necessary to maintain the specificity of the adsorption sites. There is another intracellular phase, which lacks the structural specificity for K⁺, and which contains Na⁺, Cl^-, and K⁺ in equilibrium with the external solution. The dimensions of the free intracellular phase will vary from cell to cell, but it will be smaller in the intact animal, and will increase on soaking in normal Ringer, until it is approximately one-third of the total cellular volume. The increase in this phase may be ascribed to a decrease in the energy available to maintain the ordered phase.     

Solvent Water for Electrolytes in the Muscle Fiber of the Giant Barnacle

Seven experiments are described which permit estimation of the "solvent water" or the "osmotically active water" of the dissected fiber from the giant barnacle, Balanus nubilus. Each of the first four experiments includes the measurement of a free ion activity in the myoplasm by means of a Na⁺, K⁺, or Cl^- ion-specific microelectrode. The fifth experiment makes use of a membrane potential vs. [K]_o curve. The last two experiments measured fiber water and fiber volume as bath osmolarity was changed. The seven independent estimations of solvent water ranged from 0.64 to 0.72 of fiber water with a mean of 0.68. Since the extracellular space of single fibers was about 7% of fiber water, it was concluded that 25% of analyzable water was not acting as solvent for the osmotically active solutes in the myoplasm.     

The Influence of the Intracellular Potential on Potassium Uptake by Beetroot Tissue

Intracellular potentials were measured in beetroot tissue during the steady-state uptake of K⁺ from various solutions. In solutions containing bicarbonate, the membrane potential becomes up to 70 mv more negative than the estimated equilibrium potential for K⁺. The uptake of K⁺ from such solutions is correlated with variations in the potential, both when the bicarbonate concentration is changed and also when the metabolic activity of the tissue is changed by washing in water for various periods. However, the estimated permeability to K⁺ varies from 0.4 x 10^-7 to 1.5 x 10^-7 cm·sec^-1. It is postulated that the change of potential arises from the metabolic transport of HCO₃^- into the cell or H⁺ outwards, and that the associated uptake of K⁺ is partly or entirely by passive diffusion across the cell membrane. In contrast, K⁺ uptake from KCl solutions is not accompanied by any significant change in the membrane potential, which remains relatively close to the K⁺ equilibrium potential. In solutions containing both KHCO₃ and KCl, it appears that an amount of K⁺ equal to the influx of Cl^- is taken up independently of the potential, while the component of K⁺ uptake which is not balanced by Cl^- uptake is related to the potential in the manner described. These results suggest that K⁺ uptake is linked to Cl^- uptake in an electrically neutral active transport process.     

Calcium influx in skeletal muscle at rest,during activity,and during potassium contracture

Calcium influx in the sartorius muscle of the frog (Rana pipiens) has been estimated from the rate of entry of Ca⁴⁵. In the unstimulated preparation it is about equal to what has been reported for squid giant axons, but that per impulse is at least 30 times greater than in nerve fibers. The enhanced twitch when NO^-₂ replaces Cl^- in Ringer's is associated with at least a 60 per cent increase in influx during activity, whereas this anion substitution does not affect the passive influx significantly. Calcium entry during potassium contracture is even more markedly augmented than during electrical stimulation, but only at the beginning of the contracture; thus, when a brief Ca⁴⁵ exposure precedes excess K⁺ application, C⁴⁵ uptake is increased three- to fivefold over the controls not subjected to K⁺, whereas when C⁴⁵ and K⁺ are added together, no measurable increase in Ca⁴⁵ uptake occurs. These findings are in keeping with the brevity of potassium contracture in "fast (twitch)" fibers such as in sartorius muscle.     

Muscle: A Three Phase System : The partition of divalent ions across the membrane

The partition of sulfate, Ca⁺⁺, and Mg⁺⁺ across the membrane of the sartorius muscle has been studied, and the effect of various concentrations of these ions in the Ringer solution on the cellular level of Na⁺, K⁺, and Cl^- has been determined. The level of the three divalent ions in toad plasma and muscle in vivo has been assayed. Muscle was found to contain an almost undetectable amount of inorganic sulfate. Increases in the external level of these ions brought about increases in intracellular content, calculated from the found extracellular space as determined with radioiodinated serum albumin or inulin. Less of the cell water is available to sulfate than to Cl^-, and the Mg⁺⁺ space is less than the Na⁺ space. An amount of muscle water similar to that found for Li⁺ and I^- appears to be available to these divalent ions. Sulfate efflux from the cell was extremely rapid, and it was not found possible to differentiate kinetically between intra- and extracellular material. These results are consistent with the theory of a three phase system, assuming the muscle to consist of an extracellular phase and two intracellular phases. Mg⁺⁺ and Ca⁺⁺ are adsorbed onto the ordered phase, and increments in cellular content found on raising the external level are assumed to occur in the free intracellular phase.     

The Effects of Various Ions on Resting and Spike Potentials of Barnacle Muscle Fibers

Effects of monovalent cations and some anions on the electrical properties of the barnacle muscle fiber membrane were studied when the intra- or extracellular concentrations of those ions were altered by longitudinal intra-cellular injection. The resting potential of the normal fiber decreases linearly with increase of logarithm of [K⁺]_out and the decrement for a tenfold increase in [K⁺]_out is 58 mv when the product, [K⁺]_out ·[Cl^-]_out, is kept constant. It also decreases with decreasing [K⁺]_in but is always less than expected theoretically. The deviation becomes larger as [K⁺]_in increases and the resting potential finally starts to decrease with increasing [K⁺]_in for [K⁺]_in > 250 mM. When the internal K⁺ concentration is decreased the overshoot of the spike potential increases and the time course of the spike potential becomes more prolonged. In substituting for the internal K⁺, Na⁺ and sucrose affect the resting and spike potentials similarly. Some organic cations (guanidine, choline, tris, and TMA) behave like sucrose while some other organic cations (TEA, TPA, and TBA) have a specific effect and prolong the spike potential if they are applied intracellularly or extracellularly. In all cases the active membrane potential increases linearly with the logarithm of [Ca⁺⁺]_out/[K⁺]_in and the increment is about 29 mv for tenfold increase in this ratio. The fiber membrane is permeable to Cl^- and other smaller anions (Br^- and I^-) but not to acetate^- and larger anions (citrate^-, sulfate^-, and methanesulfonate^-).     

Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout,Oncorhynchus mykiss

Summary The novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na⁺/acidic equivalent and Cl^-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na⁺ and Cl^- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO₃ infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na⁺ influx, no change in Cl^- influx, positive Na⁺ balance, negative Cl^- balance, and net H⁺ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na⁺ influx and stimulation of Cl^- influx, negative Na⁺ balance, positive Cl^- balance, and net H⁺ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na⁺ and Cl^- transport during acid-base disturbance are achieved by large increases and decreases in J_max, and by increases in K_m. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO ₃ ^- and H⁺ availability. This analysis indicated that true J_max values for Na⁺/acidic equivalent and Cl^-/basic equivalent transport are 4–5 times higher than apparent J_max values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport K_m values for HCO ₃ ^- and H⁺ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent J_max values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.Abbreviations Amm total ammonia in water - DMO 55-dimethyl-24-oxyzolidine-dione - J_in unidirectional inward ion movement across the gill - J_out unidirectional outward ion movement across the gill - J_net net transfer of ions (sum of J_in and J_out) across the gill - J_max maximal transport rate for ion - K_m inverse of affinity of transporter for ion - PIO₂ partial pressure of oxygen in inspired water - P_aCO₂ partial pressure of carbon dixide in arterial blood - TAlk titratable alkalinity of the water - PEG polyethylene glycol - NEN New England Nuclear     

Atmospheric deposition, mass balances, and processes regulating streamwater solute concentrations in mixed-conifer catchments of the Sierra Nevada, California

Solute concentrations in atmospheric deposition and stream water were measuredfrom 1984 through 1993 to determine the fate and mobility of solutes in twogauged mixed-conifer catchments (Tharp's and Log creeks) located in theSierra Nevada, California. The two catchments contain mature forest standsdominated by Abies concolor (white fir), Sequoiadendron giganteum (giantsequoia), Abies magnifica (red fir) and Pinus lambertiana (sugar pine).Ammonium, Cl^-, Ca²⁺ and NO^- ₃were highest in concentration of the solutes measured in wet deposition;bulk deposition was highest in SO^{2- 4}, NH⁺ ₄,Cl^- and H⁺. Net retention ofH⁺, NO₃ ^-, NH₄ ⁺,SO₄ ^2- and Cl^- occurred in both catchments.Discharge was dominated by spring snowmelt with the largest export yieldsfor acid neutralizing capacity (ANC), SiO₂, andCa²⁺. Export yields of H⁺,NO₃ ^-, NH₄ ⁺ and PO₄ ^3-were relatively small (0.5 kg ha^-1 y^-1).Discharge-concentration relationships for ANC, SiO₂,Na⁺, K⁺, Ca²⁺ andMg²⁺ were inverse and their concentrations in stream waterwere primarily influenced by discharge and annual differences in the relativecontributions of snowmelt and groundwater. The mobility of these solutes iscontrolled by the rates of mineral weathering and ion exchange. The positiverelationship of SO₄ ^2- concentration with increasingdischarge suggests that atmospherically deposited SO₄ ^2-is temporarily stored and that its release is controlled by the extent of soilwater flushing.     

基于典范对应分析的滨海湿地土壤季节性盐渍化特征

为了更好地开发利用黄河三角洲湿地土壤资源和生态环境建设,运用单因素方差分析(One-way ANOVA)和典范对应分析方法(CCA),对黄河口湿地0—10 cm土壤全盐量(TS)、盐分离子组成、pH、钠吸附比(SAR)、电导率(EC)的季节性变化特征及数量关系进行了分析。结果表明:该区土壤属于盐土类型,春季(5月)、夏季(8月)、秋季(10月)全盐含量均高于17.9 g/kg,且春季、秋季的含盐量高于夏季,造成春、秋季积盐,夏季脱盐。盐分阳离子以Na~+为主,阴离子以Cl~-为主,除Mg~(2+)在夏季、秋季和Cl~-在秋季表现出强烈的变异性外,其余离子在不同季节均表现出中等强度的变异性。春季,Cl~-与Na~+、Mg~(2+),SO■与K~+;夏季,Cl~-与HCO~-_3,SO■与Mg~(2+)、Ca~(2+),Ca~(2+)与Mg~(2+);秋季,Cl~-与SO■,SO■与Mg~(2+),Ca~(2+)与Mg~(2+),均有很好的关联性。pH值均介于7.7—8.1之间,各个季节受盐分离子的影响较小,分布较为均匀。SAR介于3.08—5.29之间,春季受控于HCO~-_3;夏季受控于K~+;秋季受各离子的影响均较小,分布较为均匀。EC介于7.16—13.04 mS/cm,春季受各离子的影响均较小,其空间分布较为均匀;夏季受控于SO■、Ca~(2+)、Mg~(2+);秋季受控于Na~+。TS与Cl~-、Na~+、Mg~(2+)在季节变化上的差异性一致,且各个季节均受控于Cl~-。故通过合理的措施控制或减少Cl~-来源是一条减轻黄河口湿地土壤盐渍化的合适途径。     

Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.     

油菜素内酯对盐渍下油菜幼苗生长的调控效应及其生理机制

为了探讨油菜素内酯对植物耐盐性的调控,以甘蓝型油菜"南盐油1号"为试验材料,研究了外源24-表油菜素内酯(24-EBL)对100、200 mmol/L Na Cl胁迫下油菜幼苗干重(DW)、相对含水量(RWC)、渗透调节能力(OAA)、叶片气体交换参数、气孔限制值(Ls)等的调节效应,还测定了不同器官的Na+、K+、Cl-含量,并计算各器官的K+/Na+和SK,Na。结果表明:(1)在不同浓度的盐胁迫下,油菜幼苗DW显著下降,胁迫下外源喷施10-12、10-10、10-8、10-6mol/L 24-EBL作用下,油菜植株干重均不同程度的上升,且植株干重都在10-10mol/L 24-EBL(EBL2)处理下达到最大值,分别比100、200 mmol/L Na Cl胁迫下增加29%和20%。与对照相比,非盐胁迫下外源喷施10-12、10-10、10-8、10-6mol/L 24-EBL,油菜幼苗植株干重与对照相比均无显著变化。(2)不同Na Cl浓度胁迫下,油菜叶片的RWC显著下降,外施EBL2可显著提高油菜叶片的RWC和OAA。(3)不同浓度Na Cl胁迫下,油菜幼苗叶片净光合速率(Pn)、气孔导度(Gs)、胞间CO2浓度(Ci)和蒸腾速率(Tr)均不同程度下降,而Ls显著上升,而外喷EBL2可不同程度的提高Pn、Gs、Ci、Tr,降低Ls。(4)与对照相比,Na Cl胁迫下油菜幼苗叶片、叶柄和根的Na+和Cl-含量均显著上升,Na Cl浓度愈高,Na+和Cl-含量上升愈显著。而K+含量均下降,外源EBL2可显著降低幼苗各器官的Na+和Cl-含量,对幼苗叶片K+含量没有影响,但提高了叶柄和根中的K+含量。上述表明,合适浓度的24-EBL外喷可明显提高油菜的耐盐水平,且不同浓度Na Cl胁迫下,最适24-EBL浓度均为10-10mol/L。主要是因为外源喷施24-EBL能显著改善离子稳态和渗透调节能力,从而改善盐胁迫下油菜幼苗的光合作用、水分状况,提高其耐盐性。而24-EBL对盐处理下油菜植株气孔限制的显著改善是其促进其光合、水分利用的重要原因,也是其对100 mmol/L Na Cl处理的油菜生长调控效果优于200 mmol/L Na Cl处理的重要原因之一。结果还显示,在叶片中,24-EBL外施可通过排Na+和Cl-来维持植株离子稳态,而对K+影响不大;在根、茎中可通过排Na+、排Cl-、吸K+维持稳态。     

Isolated epithelial cells of the toad bladder. Their preparation, oxygen consumption, and electrolyte content

Epithelial cells of the toad bladder were disaggregated with EDTA, trypsin, hyaluronidase, or collagenase and were then scraped free of the underlying connective tissue. In most experiments EDTA was complexed with a divalent cation before the tissue was scraped. Q _OO2, sucrose and inulin spaces, and electrolytes of the isolated cells were measured. Cells disaggregated by collagenase or hyaluronidase consumed O₂ at a rate of 4 µl hr^-1 dry wt^-1. Q _OO2 was increased 50% by ADH (100 U/liter) or by cyclic 3'',5''-AMP (10 mM/liter). Na⁺-free Ringer''s depressed the Q _OO2 by 40%. The Q _OO2 of cells prepared by trypsin treatment or by two EDTA methods was depressed by Na⁺-free Ringer''s but was stimulated relatively little by ADH. Two other EDTA protocols produced cells that did not respond to Na⁺ lack or ADH. The intracellular Na⁺ and K⁺ concentrations of collagenase-disaggregated cells were 32 and 117 mEq/kg cell H₂O, respectively. Cation concentrations of hyaluronidase cells were similar, but cells that did not respond to ADH had higher intracellular Na⁺ concentrations. Cells unresponsive to ADH and Na⁺ lack had high sucrose spaces and low transcellular membrane gradients of Na⁺, K⁺, and Cl^-. The results suggest that trypsin and EDTA disaggregation damage the active Na⁺ transport system of the isolated cell. Certain EDTA techniques may also produce a general increase in permeability. Collagenase and hyaluronidase cells appear to function normally.     

渗透胁迫和离子毒害对转Bt基因棉幼苗生长及离子分布的影响

研究了渗透和盐胁迫处理对转Bt基因抗虫棉(Gossypium hirsutum) 99B种子的萌发和幼苗生长的影响,以及幼苗不同器官离子吸收和分配的差异。结果表明:渗透和盐胁迫均对转Bt基因抗虫棉幼苗的生长有抑制作用,其中PEG的抑制作用最强,而3种盐的抑制程度以CaCl₂>NaCl>Na₂SO₄,且在Na⁺含量相同时,Cl^-的毒害大于SO₄^2-。渗透胁迫下使根、茎和叶中的Na⁺和Cl^-含量提高,K⁺、Ca²⁺、SO₄^2-含量和K⁺/Na⁺、Ca²⁺/Na⁺和SO₄^2-/Cl^-比值降低,且地上部的变化幅度大于地下部的,其中以PEG的影响最为显著,其次是CaCl₂,Na₂SO₄处理最弱。这些说明,转Bt基因抗虫棉99B的耐盐性较弱。     

Intracellular ionic compartmentation, electrical membrane properties, and cell membrane permeability before and during first cleavage in the Ambystoma egg.

The intracellular ionic distribution in uncleaved and cleaving Ambystoma eggs was investigated by analysing the influx of ³H₂O, by determining the total content of Na⁺, K⁺ and Cl^? in extracts of eggs at different stages by both flame spectrophotometry and ion-selective microelectrodes, and by the continuous measurement of the Na⁺, K⁺ and Cl^? activities (a_Naⁱ, a_Kⁱ and a_Clⁱ) using intracellular ion-selective microelectrodes. The electrical membrane potential (E_m) and membrane resistance (R_m) were measured continuously in uncleaved and normally cleaving eggs as well as in eggs cleaving after removal of the vitelline membrane. The latter eggs expose their newly formed cleavage membrane to the external medium. Ionic permeability of the cell membrane before and during cleavage was analysed by a statistical comparison of the experimentally determined relationship between E_m and the ionic gradients across the cell membrane with those predicted theoretically from a constant field equation in dependence on the relative permeability, through insertion of the measured intracellular ion activities.³H₂O influx revealed the existence of a single intracellular water compartment (3.06 μl/egg) and a low water permeability (5.35 × 10^?5 cm sec^?1). Na⁺, K⁺ and Cl^? concentrations were constant at 54.1, 72.1 and 73.1 mM respectively, while a_Naⁱ, a_Kⁱ and a_Clⁱ were constant at 5.8, 51.8 and 59.7 mM respectively. It was concluded that all Cl^? ions are in solution, while 12.5% of all K⁺ and 86% of all Na⁺ is bound. The uncleaved egg showed a positive E_m of ca 40 mV and a specific membrane resistance of 39 kOhm cm². E_m could be described by a constant field equation with a permeability ratio $\text{P}{}_{\mathrm{<mtext>K</mtext>}}\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0.073}$. Shortly after the onset of first cleavage, E_m rapidly decreased concomitant with a rise in R_m (68.5 kOhm cm²). This was interpreted as a drop in Na⁺ permeability. During the cleavage process E_m progressively hyperpolarized and R_m decreased due to the insertion of a small fraction (3.3%) of the newly formed intercellular membrane into the cleavage furrow. This new membrane had a low specific resistance (0.69 kOhm cm²). Both in normally cleaving eggs and in eggs cleaving in the absence of the vitelline membrane E_m behaved according to the constant field equation, $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}\text{P}{}_{\mathrm{<mtext>K</mtext>}}$ being 0.69 and 0.39, respectively. The differences with other amphibian eggs were discussed.     

Monovalent Cations Induce Microsporidian Spore Germination In Vitro

ABSTRACT The relative capacity of Na⁺, K⁺ and Cl^- to stimulate germination of spores of the microsporidian Nosema algerae, a pathogen of mosquitoes, was examined by ion substitution experiments. Sodium at 0.1 M was ineffective to produce the high percentage of germination that typically occurs with 0.1 M NaCl (the normal stimulation solution) if Cl^- was substituted with the usually impermeant anions SO₄^2-, HPO₄^2-, or the organic acids oxalate, cacodylate, EGTA, MES and HEPES. However, substantial concentration- and pH-dependent germination was seen with Na₂SO₄ in the 0.2-0.8 M Na⁺ range. Similar results were obtained with solutions of K⁺ accompanied by impermeant anions. In contrast, the chloride salts of usually impermeant cations, like choline and triethanolamine, failed to germinate spores even at 0.8 M unless Na⁺ or K⁺ was independently added. The presence of 0.5 M choline chloride in the medium reduced the levels of Na₂SO₄ required to produce germination down to equivalence with those of Na⁺ in the normal stimulation solution. Monensin, a Na⁺ ionophore, facilitated the germination induced by a medium-level stimulus (0.04 M NaCl) in sonicated samples. These findings indicate that N. algerae spores germinate in response to the alkali metal cations, while CI^- plays a passive role by diffusing to maintain internal electroneutrality during cation influx. A possible mechanism of cation action in spore germination is suggested on the basis of these results and observations on other systems of intracellular motility.     

Intracellular Cl? Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl⁻. The purpose of the present study was to determine (a) whether Cl⁻ plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl⁻]_i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl⁻- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na⁺ and 0–194 mM Cl⁻. The artificial seawater bathing the BMF initially contained no Na⁺. After dialysis was halted, adding 50 mM Na⁺ to the artificial seawater caused an amiloride-sensitive pH_i increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J _Na-H) increased with increasing [Cl⁻]_i under both normo- and hypertonic conditions, with similar apparent K _m values (∼120 mM). However, the maximal J _Na-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pH_i requires Cl⁻ under both normo- and hypertonic conditions, but at any given [Cl⁻]_i, J _Na-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl⁻]_i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl⁻-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl⁻-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl⁻, dialyzing with GTPγS or AlF₃, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl⁻-depleted fibers, the Cl⁻-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF₃ indicates that the G protein is a heterotrimeric G protein.     

The Membrane Potential of Acetabularia mediterranea

The cytoplasm of an Acetabularia cell is normally at a potential of about -170 mv relative to the external solution; the vacuole is also at this potential. Although there is strict flux equilibrium for all ions, the potential is more negative than the Nernst potentials of any of the permeating ions. Darkness, CCCP, low temperature, and reducing [Cl^-]_o by a factor of 25 all rapidly depolarize the membrane and inhibit Cl^- influx. Some of these treatments do not inhibit the effluxes of K⁺ and Na⁺. Increasing [K⁺]_o also depolarizes the membrane both under normal conditions and at low temperature; in the latter case the membrane is partially depolarized in normal seawater (low [K⁺]_o) and in high [K⁺]_o positive potentials of up to +15 mv are attained. It is concluded that the membrane potential is controlled by the electrogenic influx of Cl^-, and also, at least in some circumstances, by the diffusion of K⁺. In addition, it is suggested that electrogenic efflux of H⁺ may be important in transient nonequilibrium situations. An Appendix deals with the interpretation of simple nonsteady-state tracer kinetic data.     

The Nature of the In Vivo Sodium and Chloride Uptake Mechanisms through the Epithelium of the Chilean Frog Calyptocephalella gayi (Dum. et Bibr., 1841) : Exchanges of hydrogen against sodium and of bicarbonate against chloride

The Chilean frog, Calyptocephallela gayi, placed in dilute NaCl solutions may pump Na⁺ and Cl^- at very different rates depending on the kind of bath solutions in which it was preadapted. Furthermore, Na⁺ and Cl^- may be absorbed from solutions in which the accompanying coion, such as sulfate and choline, respectively, is impermeant. In all these cases it is obligatory to postulate the existence of two ionic exchange mechanisms, Cl^- and Na⁺, being exchanged against endogenous anions and cations, respectively. It has been determined that Na⁺ is exchanged against endogenous H⁺ and that Cl^- is exchanged against HCO₃^-. In animals pumping Na⁺ and Cl^- from dilute NaCl solutions Na⁺ or Cl^- uptake may be selectively inhibited, while the flux of the accompanying ion remains unchanged. This is considered to be an additional proof that both Na⁺ and Cl^- fluxes are always independent. The role of the ionic exchange mechanisms in the direct regulation of the Na⁺ and Cl^- levels in the internal medium is discussed as well as their relationship in the regulation of the acid-base equilibrium; other physioecological considerations have been treated.     

Decreased K+ conductance produced by Ba++ in frog sartorius fibers

The action of Ba⁺⁺ on membrane potential (E_m) and resistance (R_m) of frog (R. pipiens) sartorius fibers was studied. In normal Cl^- Ringer''s, Ba⁺⁺ (<9 mM) did not depolarize or induce contractions, but increased R_m slightly above the control value of 3.8 ± 0.6 KΩ-cm². In Cl^--free Ringer''s (methane sulfonate) R_m was 28.8 ± 2.8 KΩ-cm², and low concentrations of Ba⁺⁺ (0.05–5.0 mM) depolarized and induced spontaneous contractions (fibrillation), even in tetrodotoxin. To stop disturbance of the microelectrodes, contractions were prevented by using two Cl^--free solutions: (a) twice hypertonic with sucrose (230 mM), or (b) high K⁺ (83 mM) partially replacing Na⁺. In the hypertonic solution, the fiber diameters decreased, E_m increased slightly, and R_m decreased to 9.0 ± 0.6 KΩ-cm² (perhaps due to swelling of sarcotubules). Ba⁺⁺ (0.5 mM) rapidly increased R_m to 31.3 ± 3.8, decreased E_m (e.g., to -30 mv), and induced spontaneous "action potentials;" Sr⁺⁺ had no effect. In the high K⁺ solution, the fibers were nearly completely depolarized, and R_m was decreased markedly to 1.5 ± 0.2 KΩ-cm²; Ba⁺⁺ increased R_m to 6.7 ± 0.5 KΩ-cm². The Ba⁺⁺ actions usually began within 0.5 min and reached a maximum within 5 min. Addition of SO₄ ⁼, to precipitate the Ba⁺⁺, rapidly reversed the increase in R_m. Ba⁺⁺ must act by decreasing K⁺ conductance (g_K). In Cl^- Ringer''s, the high g_Cl/g_K ratio masked the effect of Ba⁺⁺ on g_K. Thus, small concentrations of Ba⁺⁺ specifically and rapidly decrease g_K.