Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin

Estimates of cytoplasmic Ca2+ concentration ([Ca2+]i) were made essentially simultaneously in the same intact frog skeletal muscle fibers with aequorin and with Ca-selective microelectrodes. In healthy fibers under truly resting conditions [Ca2+]i was too low to be measured reliably with either technique. The calibration curves for both indicators were essentially flat in this range of [Ca2+], and the aequorin light signal was uniformly below the level to be expected in the total absence of Ca2+. When [Ca2+]i had been raised to a stable level below the threshold for contracture by increasing [K+]o to 12.5 mM, [Ca2+]i was 38 nM according to aequorin and 59 nM according to the Ca-selective microelectrodes. These values are not significantly different. Our estimates of [Ca2+]i are lower than most others obtained with microelectrodes, probably because the presence of aequorin in the cells allowed us to detect damaging microelectrode impalements that otherwise we would have had no reason to reject. The observation that the light emission from aequorin-injected fibers in normal Ringer solution was below the level expected from the Ca(2+)-independent luminescence of aequorin in vitro was investigated further, with the conclusion that the myoplasm contains a diffusible macromolecule (between 10 and 30 kD) that interacts with aequorin to reduce light emission in the absence of Ca2+.     

An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis

The eggs of most or all animals are thought to be activated after fertilization by a transient increase in free cytosolic Ca2+ concentration ([Ca2+]i). We have applied Ca2+-selective microelectrodes to detect such an increase in fertilized eggs of the frog, Xenopus laevis. As observed with an electrode in the animal hemisphere, [Ca2+]i increased from 0.4 to 1.2 microM over the course of 2 min after fertilization, and returned to its original value during the next 10 min. No further changes in [Ca2+]i were detected through the first cleavage division. In eggs impaled with two Ca2+ electrodes, the Ca2+ pulse was observed to travel as a wave from the animal to the vegetal hemisphere, propagating at a rate of approximately 10 microns/s across the animal hemisphere. The apparent delay between the start of the fertilization potential and initiation of the Ca2+ wave at the sperm entry site as approximately 1 min. Through these observations describe only the behavior of subcortical [Ca2+]i, we suggest that our data represent the subcortical extension of the cortical Ca2+ wave thought to trigger cortical granule exocytosis, and we present evidence that both the timing and magnitude of the Ca2+ pulse we observed are consistent with this identity. This first quantification of subcortical [Ca2+]i during fertilization indicates that the Ca2+ transient is available to regulate processes (e.g., protein synthesis) in the subcortical cytosol.     

Role of calcium ions in the control of embryogenesis of Xenopus. Changes in the subcellular distribution of calcium in early cleavage embryos after treatment with the ionophore A23187.

Treatment of stage 5 Xenopus embryos with the ionophore A23187 for only 10 min, in the absence of extracellular Mg2+ and Ca2+, causes cortical contractions and a high incidence of abnormal embryos during subsequent development. Cation analysis shows that divalent ions are not lost from the embryos, but that Ca2+ is redistributed within the subcellular fractions. Ca2+ is probably released from yolk platelets and/or pigment granules by the action of A23187, [Ca2+] rises in the cytosol, and the mitochondria attempt to take up this free Ca2+. The mitochondria concomitantly undergo characteristic ultrastructural transformations, changing towards energized-twisted and energized-zigzag conformations. A23187 allows these changes to be demonstrated in situ. Extracellular divalent cations (10(-4) M) interfere with this intracellular action of A23187. Intracellular accumulation of Na+ (by treatment with ouabain) or Li+ also causes abnormal development, probably by promoting a release of Ca2+ from the mitochondria. It is suggested (a) that all these treatments cause a rise in [Ca2+]i which interferes with normal, integrated cell division, so causing, in turn, abnormal embryogenesis, (b) that levels of [Ca2+]i are of importance in regulating cleavage, (c) that the mitochondria could well have a function in regulating [Ca2+]i during embryogenesis in Xenopus, and (d) that vegetalizing agents may well act by promoting a rise in [Ca2+]i in specific cells in the amphibian embryo.     

Intracellular free magnesium in excitable cells: its measurement and its biologic significance

As a necessary cofactor for hundreds of enzymes, intracellular Mg2+ influences a wide range of cellular functions such as transmembrane transport of other ions, glycolysis, respiration, muscle contraction, and phosphorylation of ion channels. Unlike Ca2+, Mg2+ does not seem to have a "trigger" function. However, the wide range of enzymes requiring Mg2+ to be activated suggests that Mg2+ plays a pivotal role in fine control and coordination of cell activity, determining the "set point" of hundreds of metabolic reactions. In this sense, intracellular Mg2+ might be regarded as a static rather than a dynamic regulator of cell function. Little is known about the mechanisms by which excitable and other cells keep their [Mg2+]i within narrow limits against large electrochemical gradients. Furthermore, the actual basal level of [Mg2+]i has been the subject of recent controversy. In the present paper the roles of intracellular Mg2+ on cell function as well as four current techniques for measuring [Mg2+]i are briefly reviewed. These techniques are (i) metallochromic indicators, (ii) 31P nuclear magnetic resonance, (iii) null point for plasma membrane permeabilization using the ionophore A23187 and, (iv) Mg2+-selective microelectrodes. The relative advantages and disadvantages of each of these techniques are discussed with special emphasis on Mg2+-selective microelectrodes.     

Reducing inositol lipid hydrolysis, Ins(1,4,5)P3 receptor availability, or Ca2+ gradients lengthens the duration of the cell cycle in Xenopus laevis blastomeres

We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

Neutral carrier Na+- and Ca2+-selective microelectrodes for intracellular application.

Na+- and Ca2+-selective microelectrodes were made with Simon's neutral carrier ETH 227 and ETH 1001, respectively, and their properties were studied for intracellular application. The kNaK (selectivity coefficient for Na+ with respect to K+) values of the Na+-selective microelectrodes were in the range of 0.01-0.02, which is comparable to those of recessed-tip Na+-selective glass microelectrodes. The kNaMg values of the microelectrodes were approximately 0.005 so that the interference by intracellular Mg2+ levels could be negligible. The kNaCa values were approximately 2 and the Na+-selective microelectrodes were more selective to Ca2+ than Na+. This indicates that their intracellular application requires special care to handle Ca2+ interference under certain conditions. The kNaK, kNaMg, and kNaCa values did not depend significantly on the methods used for their determination or on the ion activity levels tested. The Nicolsky equation described well the microelectrode potentials in the mixed solutions of NaCl (1-100 mM) and KCl. Potential and resistance of the microelectrodes were stable for a long period and their response time was fast. The results indicate that the Na+-selective microlectrodes are suitable for measurements of intracellular Na ion activities. Ca2+-selective microelectrode potentials at Ca2+ concentrations lower than 10(-4) M changed significantly for the first 2-3 h and then became fairly stable. The rate of the potential change was dependent on the column length of the Ca2+-selective liquid filled. Potentials of the microelectrodes varied from 10-20 mV for Ca2+ between 10(-7) and 10(-6) M concentrations, which may be the cytosolic free-Ca2+ range. With the Ca2+ concentrations greater than 10(-6) M, the microelectrodes had potential changes of approximately 30 mV or greater for a tenfold change in Ca2+ concentration. The kCaK and kCaNa values were in the ranges of 10(-5)-10(-6) and 10(-4)-10(-5), respectively. The kCaMg values were approximately 10(-7). The results show that the Ca2+-selective microelectrodes can be used for measurements of cytosolic Ca ion activities.     

Mechanisms of calcium elevation in the micromeres of sea urchin embryos

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.     

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.     

Ca2(+)-activated K+ channels from rabbit kidney medullary thick ascending limb cells expressed in Xenopus oocytes.

Ca2(+)-activated K+ channels are present in muscle, nerve, pancreas, macrophages, and renal cells. They are important in such diverse functions as neurotransmitter release, muscle excitability, pancreatic secretion, and cell volume regulation. Although much is known about the biophysics of Ca2(+)-activated K+ channels, the molecular structure, cDNA and amino acid sequences are unknown. We injected size-fractionated mRNA isolated from cultured rabbit kidney medullary thick ascending limb cells in Xenopus oocytes and observed newly expressed K+ currents using two-microelectrode voltage-clamp technique. The expressed K+ currents are Ca2+ dependent and inhibited by charybdotoxin, a specific blocker of Ca2(+)-activated K+ channels. Amplitudes of the current ranged from 30 nA to more than 1 microA at a membrane potential of +30 mV. Reversal potential of the current suggested a K(+)-selective channel. The peak activity of Ca2(+)-activated K+ channels were observed in fractions corresponding to a message RNA with size of approximately 4.5 kilobases.     

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca(2+) signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFP -mRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca(2+). Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca(2+ )signals from ~2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca(2+ )signaling during the 2448 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca(2+) imaging window by an additional 24 hours.     

Ca2+-selective microelectrodes

Ca2+-selective microelectrodes based on the synthetic neutral carrier ETH 1001 can be used for quantitative intracellular measurements of resting Ca2+-activities and of slowly changing Ca2+-levels (response time in the order of seconds). Microelectrodes with tip diameters greater than 0.3 micron show selectivities that yield a detection limit between 10(-8) and 10(-7) M Ca2+ in an intracellular background. The Ca2+-activity is obtained together with electrical parameters of the cell (e.g. cell membrane potential and membrane resistance or conductivity). Simultaneous monitoring of other ion-activities is accessible (double- or multi-barrelled microelectrodes). The Ca2+-determination is extremely local, i.e. it probably does not indicate an averaged cytosolic activity in every situation (e.g. localized transients).     

Maturation of Xenopus oocytes is not accompanied by electrode-detectable calcium changes

Ca2+-sensitive microelectrodes were used to study changes in cytoplasmic free calcium during progesterone-induced meiotic maturation in Xenopus laevis oocytes. In contrast to previous reports, no changes were detected during the maturation process, although a large activation pulse of Ca2+ was seen when in vitro matured eggs were parthenogenetically activated. The resting level of Ca2+ in the oocytes was 0.14 microM +/- 0.05.     

Phosphorylation of IP3R1 and the regulation of [Ca2+]i responses at fertilization: a role for the MAP kinase pathway.

A sperm-induced intracellular Ca2+ signal ([Ca2+]i) underlies the initiation of embryo development in most species studied to date. The inositol 1,4,5 trisphosphate receptor type 1 (IP3R1) in mammals, or its homologue in other species, is thought to mediate the majority of this Ca2+ release. IP3R1-mediated Ca2+ release is regulated during oocyte maturation such that it reaches maximal effectiveness at the time of fertilization, which, in mammalian eggs, occurs at the metaphase stage of the second meiosis (MII). Consistent with this, the [Ca2+]i oscillations associated with fertilization in these species occur most prominently during the MII stage. In this study, we have examined the molecular underpinnings of IP3R1 function in eggs. Using mouse and Xenopus eggs, we show that IP3R1 is phosphorylated during both maturation and the first cell cycle at a MPM2-detectable epitope(s), which is known to be a target of kinases controlling the cell cycle. In vitro phosphorylation studies reveal that MAPK/ERK2, one of the M-phase kinases, phosphorylates IP3R1 at at least one highly conserved site, and that its mutation abrogates IP3R1 phosphorylation in this domain. Our studies also found that activation of the MAPK/ERK pathway is required for the IP3R1 MPM2 reactivity observed in mouse eggs, and that eggs deprived of the MAPK/ERK pathway during maturation fail to mount normal [Ca2+]i oscillations in response to agonists and show compromised IP3R1 function. These findings identify IP3R1 phosphorylation by M-phase kinases as a regulatory mechanism of IP3R1 function in eggs that serves to optimize [Ca2+]i release at fertilization.     

Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracellular stores

Iontophoresis of inositol 1, 4, 5-triphosphate into frog (Xenopus laevis) eggs activated early developmental events such as membrane depolarization, cortical contraction, cortical granule exocytosis, and abortive cleavage furrow formation (pseudocleavage). Inositol 1, 4-bisphosphate also triggered these events, but only at doses approximately 100-fold higher, whereas no level of fructose-1, 6-bisphosphate tested activated eggs. Using Ca2+-selective microelectrodes, we observed that activating doses of inositol 1, 4, 5-trisphosphate triggered a Ca2+ release from intracellular stores that was indistinguishable from that previously observed at fertilization (Busa, W. B., and R. Nuccitelli, 1985, J. Cell Biol., 100:1325-1329), whereas subthreshold doses triggered only a localized Ca2+ release at the site of injection. The subthreshold IP3 response could be distinguished from the major Ca2+ release at activation with respect to their dose-response characteristics, relative timing, sensitivity to external Ca2+ levels, additivity, and behavior in the activated egg, suggesting that the Xenopus egg may possess two functionally distinct Ca2+ pools mobilized by different effectors. In light of these differences, we suggest a model for intracellular Ca2+ mobilization by sperm-egg interaction.     

Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified, intact egg of Xenopus laevis

Using double-barreled, Ca2(+)-sensitive microelectrodes, we have examined the characteristics of the Ca2+ release by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the various layers of Xenopus laevis eggs in which the organelles had been stratified by centrifugation. Centrifugation of living eggs stratifies the organelles yet retains them in the normal cytoplasmic milieu. The local increase in intracellular free Ca2+ in each layer was directly measured under physiological conditions using theta-tubing, double-barreled, Ca2(+)-sensitive microelectrodes in which one barrel was filled with the Ca2+ sensor and the other was filled with Ins(1,4,5)P3 for microinjection. The two tips of these electrodes were very close to each other (3 microns apart) enabling us to measure the kinetics of both the highly localized intracellular Ca2+ release and its subsequent removal in response to Ins(1,4,5)P3 injection. Upon Ins(1,4,5)P3 injection, the ER-enriched layer exhibited the largest release of Ca2+ in a dosage-dependent manner, whereas the other layers, mitochondria, lipid, and yolk, released 10-fold less Ca2+ in a dosage-independent manner. The removal of released Ca2+ took place within approximately 1 min. The sensitivity to Ins(1,4,5)P3 and the time course of intracellular Ca2+ release in the unstratified (unactivated) egg is nearly identical to that observed in the ER layer of the stratified egg. Our data suggest that the ER is the major organelle of the Ins(1,4,5)P3-sensitive Ca2+ store in the egg of Xenopus laevis.     

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.     

Calcium-sensitive photoproteins

The recombinant Ca2+ sensitive photoprotein aequorin was the first probe used to measure specifically the Ca2+ concentration, [Ca2+], inside the intracellular organelles of intact cells. Aequorin-based methods offer several advantages: (i) targeting of the probe is extremely precise, thus permitting a selective intracellular distribution; (ii) the use of wild-type and low Ca2+-affinity aequorins allows covering a large dynamic range of [Ca2+], from 10(-7) to 10(-3)M; (iii) aequorin has a low Ca2+ buffering effect and it is nearly insensitive to changes in Mg2+ or pH; (iv) it has a high signal-to-noise ratio; (v) calibration of the results in [Ca2+] is made straightforward using a simple algorithm; and (vi) the equipment required for luminescence measurements in cell populations is simple and low-cost. On the negative side, this technique has also some disadvantages: (i) the relatively low amount of emitted light makes difficult performing single-cell imaging studies; (ii) reconstitution of aequorin with coelenterazine is necessary to generate the functional photoprotein and this procedure requires at least 1h; (iii) in the case of aequorin targeted to high Ca2+ compartments, because of the high rate of aequorin consumption at steady-state, only relatively brief experiments can be performed and, because of the steepness of the Ca2+-response curve, the calibrated [Ca2+] values may not reflect the real mean in cells or compartments with dyshomogeneous behavior; and (iv) expression of targeted aequorins requires previous transfection or infection to introduce the appropriate DNA construct, or alternatively the use of stable cell clones.     

Electrophysiologic parameters of ion transport systems in early developmental stages of fish and amphibia]

It is shown that the membrane potential level, ionic current and membrane conductance depend on the cell cycle stage both in Misgurnus fossilis L. embryos and in Xenopus laevis Daudin embryos by the microelectrode technics. Allowing for the role of the adenylate cyclase system in the membrane potential oscillations and ion conductance of membranes, some series of experiments for analysis of the inhibitor have been carried out.