Evidence for high molecular weight Na−Ca exchange in cardiac sarcolemmal vesicles

Summary Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na–Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na–Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (M _r) of approximately 226,000±20,000sd (n=6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 11 with mobile phase buffer containing 50mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na–Ca exchange activity. Na–Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na–Ca exchange activity were 91±7 and 68±11%, respectively. In the peak fraction, Na–Ca exchange specific activity increased two-to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimumM _r of 224,000 Da. Specific⁴⁵Ca²⁺-binding SL proteins withM _r of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose.These data suggest that native cardiac Na–Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na–Ca exchange protein(s) remains incomplete.     

Cytoplasmic free calcium in Riccia fluitans L. and Zea mays L.: Interaction of Ca2+ and pH?

In cells of Zea mays (root hairs, coleoptiles) and Riccia fluitans (rhizoids, thalli) intracellular Ca²⁺ and pH have been measured with double-barrelled microelectrodes. Free Ca²⁺ activities of 109–187 nM (Riccia rhizoids), 94–160 nM (Riccia thalli), 145–231 nM (Zea root hairs), 84–143 nM (Zea coleoptiles) were found, and therefore identified as cytoplasmic. In a few cases (Riccia rhizoids), free Ca²⁺ was in the lower millimolar range (2.3±0.8 mM). A change in external Ca²⁺ from 0.1 to 10 mM caused an initial and short transient increase in cytoplasmic free Ca²⁺ which finally levelled off at about 0.2 pCa unit below the control, whereas in the presence of cyanide the Ca²⁺ activity returned to the control level. It is suggested that this behaviour is indicative of active cellular Ca²⁺ regulation, and since it is energy-dependent, may involve a Ca²⁺-ATPase. Acidification of the cytoplasmic pH and alkalinization of the vacuolar pH lead to a simultaneous increase in cytoplasmic free Ca²⁺, while alkalinization of pH_c decreased the Ca²⁺ activity. Since this is true for such remote organisms as Riccia and Zea, it may be concluded that regulation of cytoplasmic pH and free Ca²⁺ are interrelated. It is further concluded that double-barrelled microelectrodes are useful tools for investigations of intracellular ion activities in plant cells.Symbols and abbreviations _m, _m membrane potential difference, changes thereof - PVC polyvinylchloride     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Phytochrome modulation of calcium fluxes in wheat (Triticum aestivum L.) protoplasts

Employing the metallochromic dye murexide and by monitoring the uptake of radiolabelled calcium, photoreversible calcium fluxes were measured in wheat leaf protoplast suspensions. Results obtained by both methods were identical — red light promoted and subsequent far-red irradiation reversed an influx of Ca⁺⁺ ions into the protoplasts. These findings imply phytochrome regulation of Ca⁺⁺ fluxes across the plasma membrane. The influx of Ca⁺⁺ stimulated by 2 min red irradiation could be maintained in total darkness for the initial 16–18 min after illumination, after which a 6–8 min efflux process was triggered and the basal Ca⁺⁺ level restored. Verapamil, a calcium channel blocker, inhibited the red-promoted influx, whereas the far-red mediated efflux could be checked by the use of the ATPase inhibitor vanadate, and also by the calmodulin antagonist chlorpromazine, thus suggesting a role of ion channels and pumps in phytochrome-controlled Ca⁺⁺ fluxes. The possible involvement of phosphoinositides in phytochrome-modulated calcium fluxes was also investigated.Abbreviations A difference in absorbance - CPZ chlorpromazine - FR far-red (light) - MX murexide - PI phosphatidylinositol - PIP₂ phosphatidylinositol 4, 5-bisphosphate - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - POPOP 1, 4-bis [2-(5-phenyl-1, 3-oxazolyl)]-benzene - PPO 2, 5-diphenyl-1, 3-oxazole - R red (light) - SOV sodium orthovanadate     

Effect of cholesterol and dipalmitoyl phosphatidylcholine enrichment on the kinetics of Na−Li exchange of human erythrocytes

Summary The effects of cholesterol loading and depletion and of a 10% replacement of native phosphatidylcholine by dipalmitoyl phosphatidylcholine (di 16:0-PC) on kinetic properties of human red cell Na–Li exchange have been studied.Compared to control erythrocytes (cholesterol/phospholipid ratio (C/P=0.8–0.9)),V _max of phloretin-sensitive Li uptake and of Li efflux stimulated by extracellular Na (Na _o ) were reduced by 15–30% in cholesterol-loaded red cells (C/P=1.05–1.33). The apparentK _m values for external Li (Li _o ) and for internal Li (Li _i ) were decreased by about one-third in these cells. Cholesterol depletion (C/P=0.7) exerted opposite effects on the kinetics of Na _o -dependent Li efflux. On augmentingC/P from 0.66 to 1.0,V _max of Na _o -dependent Li efflux was reduced by about 30%; increasingC/P above 1.0 caused no further lowering ofV _max. Li leakage rates monotonically decreased over the whole range ofC/P ratios examined (0.66–1.3). This indicates that Na–Li exchange and Li leak are differentially affected by cholesterol.Incorporation of di 16:0-PC (replacement of 3% of total red cell phospholipids) caused similar kinetic alterations of Na–Li exchange as a rise in membrane cholesterol by 20–50%. Notably, selective incorporation of di 16:0-PC into the outer monolayer increased both intra- and extracellular Li binding affinities of Na–Li exchange and lowered its maximum velocity. Thus, both di 16:0-PC enrichment and cholesterol loading exerted an uncompetitive type of transport inhibition. The results are in agreement with the hypothesis that the kinetic alterations of red cell Na–Li exchange seen in a subgroup of essential hypertensive patients could be due to subtle changes in the molecular species composition of individual phospholipids.     

Outward sodium and potassium cotransport in human red cells

Summary This paper reports some kinetic properties of Na–K cotransport in human red cells. All fluxes were measured in the presence of 10^–4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl₂-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na–K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na–K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr^–1 and 0.014±0.002 hr^–1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.Charge de Recherches CNRS.     

Second-messenger-induced signalling events in pollen tubes of Papaver rhoeas

A role for cytosolic free Ca²⁺ (Ca²⁺_i) in the regulation of growth of Papaver rhoeas pollen tubes during the self-incompatibility response has recently been demonstrated [Franklin-Tong et al. Plant J. 4:163–177 (1993); Franklin-Tong et al. Plant J. 8:299–307 (1995); Franklin-Tong et al. submitted to Plant J.]. We have investigated the possibility that Ca²⁺_i is more generally involved in the regulation of pollen tube growth using confocal laser scanning microscopy (CLSM). Data obtained using Ca²⁺ imaging, in conjunction with photolytic release of caged inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], point to a central role of the phosphoinositide signal transduction pathway in the control of Ca²⁺ fluxes and control of pollen tube growth. These experiments further revealed that increases in cytosolic levels of Ins(1,4,5)P₃ resulted in the formation of distinct Ca²⁺ waves. Experiments using the pharmacological agents heparin, neomycin and mastoparan further indicated that Ca²⁺ waves are propagated, at least in part, by Ins(1,4,5)P₃-induced Ca²⁺ release rather than by simple diffusion or by “classic” Ca²⁺-induced Ca²⁺ release mechanisms. We also have data which suggest that Ca²⁺ waves and oscillations may be induced by photolytic release of caged Ca²⁺. Ratio-imaging has enabled us to identify an apical oscillating Ca²⁺ gradient in growing pollen tubes, which may regulate normal pollen tube growth. We also present evidence for the involvement of Ca²⁺ waves in mediating the self-incompatibility response. Our data suggest that changes in Ca²⁺_i and alterations in growth rate/patterns are likely to be closely correlated and may be causally linked to events such as Ca²⁺-induced, or Ins(1,4,5)P₃-induced wave formation and apical Ca²⁺ oscillations.Presented at the 1997 SEB Annual Meeting: Interactive MultiMedia Biology - Experimental Biology Online Symposium, Canterbury, 7-11 April     

Effects of gibberellic acid and environmental factors on cytosolic calcium in wheat aleurone cells

Gibberellins (GAs) control a wide range of physiological functions in plants from germination to flowering. The cellular mechanisms by which gibberellic acid (GA₃) acts have been most extensively studied in the cereal aleurone. In this tissue, alterations in cellular calcium are known to be important for the primary response to GA, which is the production and secretion of hydrolytic enzymes. The extent to which cytosolic Ca²⁺ mediates the early events in GA action, however, is not known. In order to address this question, changes in cytosolic Ca²⁺ in wheat (Triticum aestivum L. cv. Inia) aleurone cells that occur rapidly after treatment with GA were characterized. In addition, GA-induced changes were compared with changes induced by three environmental stimuli that are known to modify the GA response: osmotic stress, salt (NaCl), and hypoxia. The Ca²⁺-sensitive dye fluo-3 was used to photometrically measure cytosolic Ca²⁺. It was found that GA₃ induced a steady-state increase in cytosolic Ca²⁺ of 100–500 nM. This increase was initiated within a few minutes of treatment with GA and was fully developed after 30–90 min. The changes in cytosolic Ca²⁺ that were induced by GA were distinct from those induced by mannitol, NaCl, or hypoxia. Mannitol caused a steady-state decrease whereas NaCl and hypoxia both increased cytosolic Ca²⁺. In the case of NaCl this increase was transient but for hypoxia the increase was prolonged as long as hypoxic conditions were maintained. Gibberellin-induced changes in cytosolic Ca²⁺ were not induced by the inactive GA, GA₈, nor did the GA-insensitive wheat mutant, D6899, respond to active GA₃ with altered cytosolic Ca²⁺. It is concluded that changes in cytosolic Ca²⁺ are an early and integral part of the GA response in aleurone cells. The data also indicate, however, that changes in Ca²⁺ are not sufficient, by themselves, to induce the GA response of aleurone cells.Abbreviations AM acetoxymethyl ester - GA gibberellin - GA₃ gibberellic acid - Mes 2-[N-morpholino]ethanesulfonic acid - PM plasma membrane The author is very grateful to Dr. T-h. D. Ho for his gift of D6899 grain and to Dr. R. Hooley for supplying the inactive GA₈. This work was supported by National Science Foundation Grant DCB-9206692.     

“Calcium metabolism in intact isolated thymocytes”

Summary Isolated rat thymocytes incubated under proper metabolic conditions extrude Ca²⁴ previously taken up under metabolically unfavourable conditions.The extrusion can be supported by both respiratory and glycolytic energy but glycolysis seems to be more efficient for this purpose.La^3– (50–200 M) and the ionophore A 23187 inhibit cell Ca²⁺ extrusion.Ruthenium Red (1–100 M)) does not influence cell Ca²⁺ extrusion while it inhibits the in situ mitochondrial cation uptake.All the results are consistent with a cell regulation model of Ca ²⁺ content in which both plasma membrane and mitochondria co-operate, acting in opposite directions, in order to decrease cytosolic Ca ²⁺ concentration.The possibility of Na⁺-Ca²⁺ hetero-exchange participation to cell Ca²⁺ homeostasis regulation is also discussed.     

Determination of cytoplasmic calcium concentration inDryopteris spores

Germination of Dryopteris spores is mediated by the physiologically active, far-red-absorbing form of phytochrome, Pfr, and external Ca²⁺ is necessary for the transduction of the light signal. Because knowledge about the cytoplasmic calcium ion concentration, [Ca²⁺]_i, is of great importance for understanding the role of calcium during signal transduction, this value was measured using fura-2 in fern spores undergoing the normal developmental progression into germination. Fura-2 was loaded into the spores by electroporation, which does not disrupt the normal process of germination. The intensity of the fluorescence emission of the loaded fura-2 was analysed by a microspectrophotometric assay of single spores, and successful loading could be obtained by the application of ten electrical pulses (field strength 7.5 kV · cm^–1, half-life (time constant) 230 s). Fura-2 was alternately excited by light of wavelengths 355 and 385 nm through an inverted fluorescence microscope, and the emitted fura-2 fluorescence was collected by a silicon-intensified video camera. The cytoplasmic calcium ion concentration was calculated from the ratio of the camera output obtained for both wavelengths and displayed by a pseudo-color technique. Spores responded to changes of the extracellular Ca²⁺ concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm. The substitution of a low external [Ca²⁺] (1 mM ethyleneglycol-bis(2-aminoethyl-ether) {ie166-01},N-tetraacetic acid (EGTA)) by 1 mM CaCl₂ caused a fast increase of [Ca²⁺]_i from approx. 50 nM to above 500 nM. In contrast, the subsequent substitution of CaCl₂ by EGTA decreased [Ca²⁺]_i again below 100 nM within 0.5 h. Furthermore, the application of ionomycin could initiate a change in [Ca2+]_i according to the Ca²⁺ gradient established between the extracellular medium and cytoplasm. In spores sown on a Ca²⁺-free medium, [Ca²⁺]_i, analysed in a buffer containing EGTA, was found to be around 50 nM during the first days of cultivation, independent of the irradiation protocol. However, if spores were grown in darkness on a Ca²⁺-containing medium and analysed in EGTA, [Ca²⁺]_i was significantly higher ( 500 nM). In red-light-irradiated spores, [Ca²⁺]_i was found to decrease with increasing time after irradiation, and was determined to be less than 100 nM when analysis was done 44 h after germination was initiated by the light treatment.Dedicated to Professor H. Mohr on the occasion of his 60th birthday     

Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

Imaging cytosolic free-calcium distribution and oscillations in pollen tubes with confocal microscopy: A comparison of different dyes and loading methods

Summary A steep, oscillating tip-focused gradient in cytosolic free calcium ([Ca²⁺]_c) has been implicated in pollen tube growth. Further understanding of the biological causes and consequences of these processes relies on the precise imaging of [Ca²⁺]_c during the different growth phases. In this work, the minimum technical requirements for confocal [Ca²⁺]_c imaging ofAgapanthus umbellatus pollen tubes were examined. A range of dyes, dye forms, and loading methods were compared. Non-ratio and ratio imaging were critically analysed, in terms of the detection of the [Ca²⁺]_c gradient and its fluctuations over time. Both ratiometric and nonratiornetric methods detected relative changes in [Ca²⁺]_c. However, visualisation of the [Ca²⁺]_c gradient, with an accurate spatial definition, was only possible with ratiometric methods. The gradient observed in this study ranged from 1.8 M (tip) to 180–220 nM (basal level), within the first 4–10 m. Apical [Ca²⁺]_c fluctuations with an amplitude between 415 nM and 1.8 M showed a period of 40 to 75 s. All protocols for dye-loading proved to have strengths and weaknesses. Thus, the choice of a dye and its loading procedure should consider the required imaging period, extent of sequestration, effect on cell performance and viability, ease of loading procedure, and aim of the study. The present study constitutes an examination of the [Ca²⁺]_c gradient in pollen tubes by these criteria.Abbreviations CLSM confocal laser scanning microscope - [Ca²⁺]_c cytosolic free calcium - PT pollen tube Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Ouabain-dependent potassium-potassium exchange in the toad bladder

Summary Recent results from this laboratory have indicated the existence of two potassium compartments in the isolated toad bladder. Only one of these, containing less than 10% of total intracellular potassium, appears to be related to the sodium transport system, since potassium influx at the serosal border of this compartment is coupled to the sodium efflux which occurs there. Ouabain, which specifically inhibits serosal sodium exit, has no effect on potassium fluxes and compartment sizes in bladders mounted in normal (2.5mm K) Ringer's solution. However, in the presence of this inhibitor, removal of serosal potassium results in a significant decrease in the rate coefficient for potassium efflux into the serosal medium, while an increase in serosal potassium results in a significant rise in this parameter, which appears to saturate at approximately 5mm K. This sensitivity to serosal potassium is seen neither in the absence of ouabain nor when the sodium pump is inactivated by removal of sodium from the mucosal medium. Furosemide, which also inhibits the sodium transport system, both inhibits potassium transport parameters in normal Ringer's and abolishes the potassium-sensitive potassium efflux seen in the presence of ouabain. Thus, the Na–K pump appears to operate as a K–K exchanger when the sodium system is inhibited by ouabain; this K–K exchange mechanism is inhibited by furosemide. One explanation for these results is that ouabain effects an alteration in the affinities of the transport system for sodium and potassium.     

Implication of the nucleus in excitation contraction coupling of heart cells

In the present study, Fluo-3 Ca²⁺ measurement and confocal microscopy techniques were used in order to localize cytosolic [ ]_c and nuclear [ ]_n free Ca²⁺ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca²⁺ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca²⁺ from 100–1600 nM gradually increased [Ca²⁺]_n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca²⁺ and the uptake and release of Ca²⁺ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca²⁺ levels remained elevated for several milliseconds even after cytosolic Ca²⁺ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca²⁺ concentration during excitation-contraction coupling in heart cells.     

Stoichiometry and ion affinities of the Na−K−Cl cotransport system in the intestine of the winter flounder (Pseudopleuronectes americanus)

Summary Na–K–Cl cotransport stoichiometry and affinities for Na, K and Cl were determined in flounder intestine. Measurement of simultaneous NaCl and RbCl influxes resulted in ratios of 2.2 for Cl/Na and 1.8 for Cl/Rb. The effect of Na and Rb on Rb influx showed first order kinetics withK _1/2 values of 5 and 4.5mm and Hill coefficients of 0.9 and 1.2, respectively. The effect of Cl on rubidium influx showed a sigmoidal relationship withK _1/2 of 20mm and a Hill coefficient of 2.0. The effects of variations in Na and Cl concentration on short-circuit current (I _sc) were also determined. TheK _1/2 for Na was 7mm with a Hill coefficient of 0.9 and theK _1/2 for Cl was 46mm with a Hill coefficient of 1.9. Based on the simultaneous influx measurements, a cotransport stoichiometry of 1Na1K2Cl is concluded. The Hill coefficients for Cl suggest a high degree of cooperativity between Cl binding sites. Measurements of the ratio of net Na and Cl transepithelial fluxes under short-circuit conditions (using a low Na Ringer solution to minimize the passive Na flux) indicate that the Cl/Na flux ratio is approximately 21. Therefore Na recycling from serosa to mucosa does not significantly contribute to theI _sc. Addition of serosal ouabain (100 m) inhibited Rb influx, indicating that Na–K–Cl cotransport is inhibited by ouabain. This finding suggests that a feedback mechanism exists between the Na–K-ATPase on the basolateral membrane and the apical Na–K–2Cl cotransporter.     

Auxin causes oscillations of cytosolic free calcium and pH inZea mays coleoptiles

In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pH_c), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pH_c/, _m, Ca²⁺/ _m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3–7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations in _m, pH_c and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. The _m oscillations are attributed to changes in activity of the H⁺-extrusion pump at the plasmalemma, triggered off by pH and controlled by pH regulation (pH oscillation). The origin of the pH_c and Ca²⁺ changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca²⁺ changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.Abbreviations and symbols FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - pH_c cytosolic pH - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - _m membrane potential difference (mV)     

Regulatory changes of membrane transport and ouabain binding during progesterone-induced maturation ofXenopus oocytes

Summary During progesterone-stimulated maturation of defolliculated full-grownXenopus oocytes, the activities of the transport systems forl-alanine, thymidine, chloride, phosphate, and alkali ions decrease. Differences of the extent and time course of these changes suggest that they are controlled by at least partially independent mechanisms.A closer investigation of the Na-K ATPase has shown that in unstimulated oocytes, ouabain produces maximal inhibition when 8–12×10⁹ molecules are bound per cell. This number is bound during the first phase of a diphasic uptake process. Since this phase can be suppressed by increasing the concentration of external K⁺ to 45 mmol/liter or more, it is concluded that it refers to binding to the Na–K pump in the plasma membrane. Ouabain bound prior to progesterone-induced germinal vesicle breakdown (GVBD) remains bound after the breakdown, although the Na–K pump loses the capacity to bind ouabain after GVBD in oocytes that had not been exposed to ouabain preceding GVBD. In the presence of Mg⁺⁺ membranes isolated before regulatory inhibition of pumping and ouabain binding show a Na⁺-dependent incorporation of³²P from -[³²P]-ATP that can be reversed by the addition of K⁺. The phosphorylation site migrates on LiDS-polyacrylamide gel electropherograms at about 98,000 daltons and can be identified as a Commassie blue-stainable band.