Distribution and role of calmodulin in tip growing hyphae of Saprolegnia ferax

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Visualization of actin arrays in growing hyphae of the fungusSaprolegnia ferax

Summary We have observed the distribution of filamentous actin in growing hyphae of the oomyceteSaprolegnia ferax. The actin was stained by electroporating intact hyphae in the presence of 4×10^–8 M rhodamine phalloidin. Hyphae quickly recovered from electroporation and showed an apical cap of densely packed actin filaments. The pores created by the electric shock resealed in 8–10min and within 1/2 h hyphae resumed growth and appeared normal. This technique allows us to observe actin arrays during growth and may prove to be a useful tool in determining the complex roles of actin in apical growth.Abbreviations RP rhodamine phalloidin - F-actin filamentous actin     

Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa

Calcium and proton ion fluxes were mapped at the growing apices of two hyphal organisms, the oomycete Saprolegnia ferax and the ascomycete Neurospora crassa and pseudohyphal Saccharomyces cerevisiae using self-referencing ion-selective probes. S. ferax exhibited well-defined transport zones absent in N. crassa. Ca2+ fluxes were located within 8 microm of the growing hyphal tip; the net Ca2+ flux was either inward (75% of all experiments) or outward. The inward component of the net flux was inhibited by Gd3+, known to inhibit Ca2+ permeable stretch-activated channels. Because the Ca2+ flux is located at the region of maximal hyphal expansion, exocytosis may contribute to Ca2+ efflux, in addition to the stretch-activated channel mediated influx. Maximal inward H+ flux was observed 10-30 microm behind the hyphal tip where peak mitochondria densities taper off at the onset of a vacuolation zone, presumably due to highly localized H+ cotransporter activity. By contrast, N. crassa exhibited no net Ca2+ flux and a consistently inward H+ flux (93% of all experiments) that was homogeneously distributed up to 60 microm behind the hyphal apex. Both hyphal organisms have similar tip morphology and growth rates, and are reported to have tip-high cytosolic Ca2+ gradients associated with growth. Only S. ferax exhibited tip-localized Ca2+ fluxes and a well defined H+ influx zone just behind the tip. Differences in ecological habitats and cytology--S. ferax is an aquatic organism that grows as a migrating plug of cytoplasm while N. crassa is normally terrestrial with a cytoplasm-rich mycelium and highly active cytoplasmic streaming behind the growing margin--may account for the differences in the 'architecture' of ion transport occurring during the process of tip growth. Net Ca2+ efflux and H+ influx of growing S. cerevisiae pseudohyphae were also measured but localization was not possible due to small cell size.     

Rapid wound responses ofSaprolegnia ferax hyphae depend upon actin and Ca2+-involving deposition of callose plugs

Summary Growing hyphae of the oomyceteSaprolegnia ferax wounded by impalement with a ca. 0.2 m diameter glass microelectrode normally respond within seconds with an apically directed cytoplasmic contraction followed by production of a plug which encases the electrode and occludes its recording of transmembrane potentials. This plug contains callose and Ca²⁺-associated membranes. To characterize the rapid wounding response, we disrupted specific filamentous (F) actin populations and Ca²⁺ regulation. Plug formation is inhibited by disruption of F-actin populations and low exogenous Ca²⁺ but not by inhibition of stretch-activated Ca²⁺ channels with Gd³⁺. Therefore, stretch-activated channels are not the immediate sensor. Instead, sensing may involve strain on the actin cytoskeleton which triggers the occlusion response. This wound response is qualitatively similar to the production of septa which isolate developing sporangia and seal severed hyphae, indicating the use of a normal basic cellular developmental system as a protective mechanism against environmental damage. The wound response is essential, since an inability to seal sites of mechanical damage is potentially catastrophic in acellular coenocytic organisms.Abbreviations APW artificial pond water - BAPTA 1,2-bis(orthzo-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - CTC chlortetracycline - DIC Nomarski differential interference contrast microscopy - F-actin filamentous actin - LatB latrunculin B - PM plasma membrane - RP rhodamine-labeled phalloidin - SA channels stretch-activated channels     

Behavior of kinetochores during mitosis in the fungus Saprolegnia ferax

In rapidly growing hyphae of Saprolegnia ferax, all nuclei contain arrays of kinetochore microtubules, which suggests that the nuclei are all in various phases of mitosis, with no apparent interphase. In prophase nuclei, kinetochore microtubules form a single, hemispherical array adjacent to the centrioles. This array separates into two similar arrays after centriole replication. The two arrays form by separation of the initial group of microtubules, with no kinetochore replication. During metaphase, between 6.5 and 85% of the kinetochores occur as amphitelic pairs, with a slight tendency for pairing to increase as the spindle elongates. 100% pairing has never been observed. The interkinetochore distance in these pairs is consistently similar to or approximately 0.17 microns. Throughout metaphase and early anaphase, there is extensive and increasing diversity in kinetochore microtubule length, so that a true metaphase plate has not been found. During metaphase, anaphase, and telophase, kinetochore numbers vary considerably, with a mean of similar to or approximately 30 per half spindle. A number of artefactual causes for this variability were examined and discarded. Thus, these results are accepted as real, suggesting either variable ploidy levels in the coenocytic hyphae or kinetochore replication during mitosis.     

Cytosolic free Ca2+ concentration and intracellular calcium distribution of Ca2+-tolerant isolated heart cells

The cytosolic free Ca2+ concentration of calcium-tolerant rat myocytes has been measured by the null point titration technique using arsenazo III as a Ca2+ indicator and digitonin to permeabilize the plasma membrane. The mean value obtained for 8 separate preparations was 270 +/- 35 nM. The distribution of releasable calcium between the mitochondrial and sarcoplasmic reticular compartments was measured by the successive additions of uncoupler and A23187 to cells pretreated with ruthenium red. The relative distribution of calcium in each pool was independent of the cell calcium content up to the maximum value of releasable calcium investigated (4.5 nmol/mg of cell dry weight) and was distributed in the approximate ratio of 2:1 in favor of the sarcoplasmic reticulum. The cells contained 1 nmol of calcium/mg of cell dry weight in a form nonreleasable by A23187, which was independent of the total cell calcium content as measured by atomic absorption spectroscopy. It is calculated that the calcium content of mitochondria in heart under physiological conditions is about 5 nmol/mg of mitochondrial protein. At this level, the mitochondria are likely to provide effective buffering of the cytosolic free Ca2+ concentration of quiescent heart cells. The corresponding intramitochondrial free Ca2+ is in a range above values needed to regulate the activity of Ca2+-dependent enzymes of the citric acid cycle in heart. The physiological calcium content of the sarcoplasmic reticulum in heart cells is estimated to be about 2.5 nmol/mg of cell dry weight, which is at least 5-fold greater than the amount of calcium release calculated to cause maximum tension development of cardiac muscle.     

Plasma membrane-adjacent actin filaments, but not microtubules, are essential for both polarization and hyphal tip morphogenesis in Saprolegnia ferax and Neurospora crassa

The organization and roles of F-actin and microtubules in the maintenance and initiation of hyphal tip growth have been analyzed in Saprolegnia ferax and Neurospora crassa. In hyphae of both species, the apex is depleted of microtubules relative to subapical regions and near-normal morphogenesis occurs in concentrations of nocodazole or MBC which remove microtubules, slow growth, and disrupt nuclear positioning. In contrast, each species contains characteristic tip-high arrays of plasma membrane-adjacent F-actin, whose organization is largely unaltered by the loss of microtubules but disruption of which by latrunculin B disrupts tip morphology. Hyphal initiation and subsequent normal morphogenesis from protoplasts of both species and spores of S. ferax are independent of microtubules, but at least in S. ferax obligatorily involve the formation of F-actin caps adjacent to the hyphal tip plasma membrane. These observations indicate an obligatory role for F-actin in hyphal polarization and tip morphogenesis and only an indirect role for microtubules.     

The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth

Summary The involvement of exogenous calcium ions in the regulation of pollen tube formation has been investigated in Haemanthus albiflos L. and Oenothera biennis L. by following the changes that occur in pollen germination, tube growth, and ⁴⁵⁺Ca²⁺ uptake and distribution upon application of Verapamil (an inhibitor of calcium channels), lanthanum (a Ca²⁺ substitute), and ruthenium red (believed to raise the intracellular calcium level). It was found that exogenous Ca²⁺ takes part in the formation of the calcium gradient present in germinating pollen grains and growing pollen tubes. Ca²⁺ ions enter the cells through calcium channels. Raising or reducing ⁴⁵Ca²⁺ uptake causes disturbances in the germination of the pollen grains and in the growth of the pollen tubes.     

Effect of low extracellular Ca2+ on growth spreading area,cytoplasmic Ca2+ concentration,and intracellular pH in normal and transformed human fibroblasts

The transformation of certain cells reduces the requirement of extracellular Ca²⁺ for growth. The SV-40 transformed human lung fibroblasts, WI-38 VA13, require less Ca²⁺ than normal WI-38 cells. Spreading area of normal cells decreases when cultured in 10 μM Ca²⁺ medium. Intracellular calcium concentration ([Ca²⁺]_i), of the normal and transformed cells cultured in 10μM and 2 mM Ca²⁺ media was measured by the fluorescence microscope technique using fura-2 as a probe. The [Ca²⁺], is measured in the resting state and during mobilization by serum or bradykinin stimulation. The lowering of extracellular calcium concentration results in a decrease in the resting state [Ca²⁺],_i of both normal and transformed cells. Although the total decrease in [Ca²⁺]_i is the same for both cell, the rate of decrease is much faster in normal cells than in transformed cells. Low extracellular Ca²⁺ reduces the number of cells responsive to the serum or bradykinin stimulation and decreases the peak [Ca²⁺]_i value in both cells. In addition, we investigated, using BCECF as a fluorecent probe, the intracellular pH (pH_i) of normal and transformed cells maintained at low and normal Ca²⁺. The low Ca²⁺ condition makes pH_i acidic in normal cells but not in transformed cells. The acidification of the normal cell is accompanied by a decrease in the spreading area of the cells. The decrease of the cell attacment, followed by the reduced spreading area, induced the acidic pH_i. These results suggest that the reduced Ca²⁺ requirement of transformed cells for growth is related to the mechanism of pH_i regulation rather than Ca²⁺ homeostasis and, possibly, to the anchorage-independent growth, which is a unique feature of transformed cells. © 1993 Wiley-Liss, Inc.     

Effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa in vitro

The effects of taurine on the motility and intracellular free Ca2+ concentration of fowl spermatozoa were investigated in vitro. The addition of taurine, within the range of 0-5 mmol l(-1), did not appreciably affect the motility of intact fowl spermatozoa. Motility remained almost negligible at 40 degrees C, while vigorous movement was observed at 25 degrees C. Even with the addition of Ca2+ before the addition of taurine, neither stimulation nor inhibition of motility was observed compared with the control (no addition of taurine). Similar results were obtained by the addition of taurine and calyculin A, a specific inhibitor of protein phosphatases. There were no changes in intracellular free Ca2+ concentrations, measured by a fluorescent Ca2+ indicator, fura-2, in taurine-treated spermatozoa. These results suggest that taurine is not involved in the regulation of fowl sperm motility and metabolism by intracellular Ca2+ mobilization in vitro.     

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC.     

Measuring of intracellular Ca2+ concentration in macrophages. Effects of platelet activation factor

In experiments on mice resident and stimulated thioglycolate macrophages the changes in cytoplasmic Ca2+ concentration Ca2+ have been studied by the use of the fluorescent probe fura-2. PAF acether (10(-7) M) raised Ca2+ by 300-400 nM within 1 min only in the stimulated macrophages. In the resident cells this increase was much less. In the presence of 2mM EGTA, PAF raised Ca2+ to a lesser extent. This suggests that PAF causes influx of exogenous Ca2+ through the receptor-mediated channels as well as releasing Ca2+ from intracellular stores.     

Roles of calcium ions in hyphal tip growth.

A role for Ca2+ in the tip growth process of fungal hyphae and other eukaryotic walled cells has been widely explored, following the earlier indications of their importance by Jaffe, Steer, and their colleagues. Analysis of the literature on fungi, with selected comparison with other tip-growing plant cells, shows that the growth rate and morphology of hyphae are sensitive to factors which influence intracellular Ca2+. These factors include variations in extracellular Ca2+ concentrations, Ca2+ ionophores, inhibitors of Ca2+ transport, and calmodulin- and Ca(2+)-binding dyes and buffers introduced into the cytoplasm. The effects of these agents appear to be mediated by a tip-high gradient of cytoplasmic free Ca2+ which is obligatorily present in all critically examined growing tips. Most recent observations agree that the gradient is very steep, declining rapidly within 10 to 20 microns of the tip. This gradient seems to be generated by the combined effects of an influx of Ca2+, via plasma membrane, possibly stretch-activated, channels localized in the hyphal tip, and subapical expulsion or sequestration of these ions. Expulsion probably involves a plasma membrane Ca(2+)-ATPase, but it is not yet possible to differentiate among mitochondria, endoplasmic reticulum, or vacuoles as the dominant sites of sequestration. It is suggested that regulation of the Ca2+ gradient in turn modulates the properties of the actin-based component of the cytoskeleton, which then controls the extensibility, and, possibly, the synthesis of the hyphal apex. Regulatory feedback mechanisms intrinsic to this model of tip growth regulation are briefly discussed, together with suggestions for future experiments which are crucial to its further elucidation and establishment.     

Effects of Equex from different sources on post-thaw survival,longevity and intracellular Ca2+ concentration of dog spermatozoa

The aims of the present study were to compare the effects of two commercial preparations (Equex STM Paste or Equex Pasta), whose active ingredient is sodium dodecyl sulphate (SDS), added to a Tris-egg yolk-based extender, on post-thaw sperm survival and longevity, as well as on the intracellular Ca(2+) concentration of dog spermatozoa during incubation at 38 degrees C. One ejaculate was collected from each of eight dogs. Each ejaculate was centrifuged, the semen plasma discarded, and the sperm pellet rediluted with a Tris-glucose-egg yolk extender containing 3% glycerol (Ext-1) at a sperm concentration of 200 x 10(6) spermatozoa (spz)/ml. The diluted semen was divided in three aliquots of equal volume and allowed to equilibrate for 1h at 4 degrees C. After equilibration, the same volume of three different second extenders was added, respectively, to each of the three aliquots: (A) Ext-2A (same composition as Ext-1 except that it contained 7% glycerol and 1% Equex STM Paste), (B) Ext-2B (same composition as that of Ext-1 except that it contained 7% glycerol and 1% Equex Pasta), and (C) Ext-2 (Control: same composition as that of Ext-1 except that it contained 7% glycerol). Semen samples were packed in 0.5 ml straws and frozen on a rack 4 cm above liquid nitrogen (LN(2)) in a styrofoam box. Thawing was at 70 degrees C for 8s. Sperm motility was evaluated after thawing and at 1 h intervals for 5h at 38 degrees C by subjective examination and by using a CASA system. Plasma membrane integrity and acrosomal status were evaluated at 1, 4 and 7h post-thaw using a triple staining procedure and flow cytometry. Intracellular Ca(2+) concentration of live spermatozoa was evaluated by flow cytometry at 1, 4 and 7h post-thaw after co-loading the sperm cells with the Ca(2+) indicators Fluo 3 AM and Fura Red AM, and with PI. Post-thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better (P<0.005) when Ext-2A (containing Equex STM Paste) was used. There was no difference between Ext-2B (containing Equex Pasta) and Ext-2 (Control). The mean intracellular Ca(2+) concentration (arbitrary units) of cryopreserved spermatozoa (range: 0.23+/-0.12 to 1.26+/-0.46) was higher than that of fresh spermatozoa (0.13+/-0.06). When using Ext-2A, the live spermatozoa frequently (P=0.012) appeared divided in two subpopulations, with high (1.26+/-0.46) and low (0.27+/-0.09) intracellular Ca(2+) content, respectively. When using Ext-2B or Ext-2, the live spermatozoa were more frequently seen in a single population with low intracellular Ca(2+) concentration (0.30+/-0.35 and 0.23+/-0.12, for Ext-2B and Ext-2, respectively).     

Na+ /Ca2+ exchanger contributes to asterosap-induced elevation of intracellular Ca2+ concentration in starfish spermatozoa

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.     

Critical intracellular Ca2+ concentration for all-or-none Ca2+ spiking in single smooth muscle cells.

Neurotransmitters induce contractions of smooth muscle cells initially by mobilizing Ca2+ from intracellular Ca2+ stores through inositol 1,4,5-trisphosphate (InsP3) receptors. Here we studied roles of the molecules involved in Ca2+ mobilization in single smooth muscle cells. A slow rise in cytoplasmic Ca2+ ([Ca2+]i) in agonist-stimulated smooth muscle cells was followed by a wave of rapid regenerative Ca2+ release as the local [Ca2+]i reached a critical concentration of approximately 160 nM. Neither feedback regulation of phospholipase C nor caffeine-sensitive Ca(2+)-induced Ca2+ release was found to be required in the regenerative Ca2+ release. These results indicate that Ca(2+)-dependent feedback control of InsP3-induced Ca2+ release plays a dominant role in the generation of the regenerative Ca2+ release. The resulting Ca2+ release in a whole cell was an all-or-none event, i.e. constant peak [Ca2+]i was attained with agonist concentrations above the threshold value. This finding suggests a possible digital mode involved in the neural control of smooth muscle contraction.     

Tributyltin increases cytosolic free Ca2+ concentration in thymocytes by mobilizing intracellular Ca2+, activating a Ca2+ entry pathway, and inhibiting Ca2+ efflux.

The immunotoxic environmental pollutant tri-n-butyltin (TBT) kills thymocytes by apoptosis through a mechanism that requires an increase in intracellular Ca2+ concentration. The addition of TBT (EC50 = 2 microM) to fura-2-loaded rat thymocytes resulted in a rapid and sustained increase in the cytosolic free Ca2+ concentration ([Ca2+]i) to greater than 1 microM. In nominally Ca(2+)-free medium, TBT slightly but consistently increased thymocyte [Ca2+]i by about 0.11 microM. The subsequent restoration of CaCl2 to the medium resulted in a sustained overshoot in [Ca2+]i; similarly, the addition of MnCl2 produced a rapid decrease in the intracellular fura-2 fluorescence in thymocytes exposed to TBT. The rates of Ca2+ and Mn2+ entry stimulated by TBT were essentially identical to the rates stimulated by 2,5-di-(tert.-butyl)-1,4-benzohydroquinone (tBuBHQ), which has previously been shown to empty the agonist-sensitive endoplasmic reticular Ca2+ store and to stimulate subsequent Ca2+ influx by a capacitative mechanism. The addition of excess [ethylenebis(oxyethylenenitrilo)]tetraacetic acid to thymocytes produced a rapid return to basal [Ca2+]i after tBuBHQ treatment but a similar rapid return to basal [Ca2+]i was not observed after TBT treatment. In addition, TBT produced a marked inhibition of both Ca2+ efflux from the cells and the plasma membrane Ca(2+)-ATPase activity. Also, TBT treatment resulted in a rapid decrease in thymocyte ATP level. Taken together, our results show that TBT increases [Ca2+]i in thymocytes by the combination of intracellular Ca2+ mobilization, stimulation of Ca2+ entry, and inhibition of the Ca2+ efflux process. Furthermore, the ability of TBT to apparently mobilize the tBuBHQ-sensitive intracellular Ca2+ store followed by Ca2+ and Mn2+ entry suggests that the TBT-induced [Ca2+]i increase involves a capacitative type of Ca2+ entry.     

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.     

Recording of intracellular Ca2+ from smooth muscle cells by sub-micron tip, double-barrelled CA2+-selective microelectrodes

Novel, double-barrelled Ca2+-selective microelectrodes with tip diameters of approximately 0.1 micron were constructed by using Simon's neutral Ca2+ ligand (ETH 1001). Concentric micropipettes were utilized for the first time for Ca2+-selective microelectrodes in which the Ca2+ ligand was incorporated into a protruding inner pipette, surrounded by an outer reference electrode. In addition, they were made from high resistance aluminosilicate glass tubing (Corning Code 1724). These Ca2+-selective electrodes had linear responses from pCa 3 to pCa 7 in the presence of constant [K+]. They provided on-line observation of changes in intracellular [Ca2+] and in the resting membrane potential in single smooth muscle cells isolated from toad stomach. The mean concentration of intracellular Ca2+ in resting cells was 163.6 +/- 20 nM (+/- SEM, n = 16). Doubling the intracellular Ca2+ level by exposure of cells to elevated [K+] was sufficient to cause shortening.