Mechanosensitive Ca2+ release from intracellular stores in Nitella flexilis

We found previously that the cytoplasmic drop isolated from internodal cells of Nitella flexilis releases Ca2+ in response to hypotonic treatment and named the phenomenon hydration-induced Ca2+ release (HICR). The HICR is assumed to be a result of activation of Ca2+ permeable channels in the membrane of Ca2+ stores in a stretch-activated manner. To prove this idea, mechanical stimulus was applied to the drop by means of shooting isotonic/hypnotic medium or silicon oil into the drop, or compressing the drop. All these mechanical stimuli induced a rapid increase in the Ca2+ concentration of the drop. The chloroplast fraction isolated from the cytoplasmic drop released Ca2+ on compression, while the chloroplast-free cytoplasm did not. In Chara corallina, the cytoplasmic drop, which shows a very weak HICR, also responded weakly to the mechanical stimulus, but the chloroplast fraction was inert. When chloroplasts from Chara were added to the chloroplast-free cytoplasm of N. flexilis, the cytoplasm recovered the mechanoresponse. Starch grains were as effective as chloroplasts. The data indicate that Ca2+ permeable channels in the membrane of Ca2+ stores in N. flexilis are really mechano-sensitive.     

Cytoplasmic Hydration Triggers a Transient Increase in Cytoplasmic Ca2+ Concentration in Nitella flexilis

Cells of Nitella flexilis were made inexcitable by treatmentwith 10 mM KCl for more than 24 h. A Ca²⁺-sensitive photoproteinaequorin was injected into the cytoplasm of such cells. Forvacuolar per fusion, the central part of an aequorin-loadedcell was immersed in silicone oil, and both cell ends bathedin the perfusion medium were cut off. A large light emissionfrom aequorin was observed when the vacuole was perfused witha hypotonic medium whose osmotic pressure was adjusted to halfof the osmotic pressure of the cell sap. This shows that hydrationof the cytoplasm triggers release of Ca²⁺ from internal stores,since influx of Ca²⁺ from silicone oil is excluded. Hydration of cells was induced in another way. Cells were firstdehydrated by transferring them from 10 mM KCl solution to thatwith 250 mM sorbitol added. This procedure did not affect thecytoplasmic streaming. When cells were rehydrated by transferringthem to 10 mM KCl solution, cytoplasmic streaming was eitherstopped or slowed down in a few seconds. A quick light emissionfrom aequorin was observed in the rehydration, evidence thatcytoplasmic streaming was inhibited by an increase in the cytoplasmicCa²⁺ concentration. Both streaming cessation and aequorin lightemission were observed even in KCl-treated cells which werefurther treated with 5 mM EGTA. Thus, the increase in Ca²⁺ isconcluded to be caused by the release of Ca²⁺ from internalstores. These results support our previous hypothesis [Tazawa et al.(1994) Plant Cell Physiol. 35:63] that, in Nitella flexilis,the increase in the concentration of Ca²⁺ in the cytoplasm whichoccurs on the endoosmotic side of the cell during transcellularosmosis is caused by hydration of the cytoplasm. (Received June 6, 1994; Accepted December 26, 1994)     

Tonoplast Action Potential of Characeae

The plasmalemma action potential was found to be indispensableto the production of the tonoplast action potential. In a solutionlacking Ca²⁺ and containing other divalent cations such as Ba²⁺,Mg²⁺ or Mn²⁺, the plasmalemma excited in Nitella but did notin Chara. In Nitella, however, both the tonoplast action potentialand EC-coupling were abolished due to depletion of Ca²⁺ fromthe external medium. Ca²⁺ ions injected into the cytoplasmiclayer caused a transient change in both plasmalemma and tonoplastpotentials. These results suggest that a transient rise in Ca²⁺concentration during excitation of the plasmalemma may triggerthe tonoplast action potential. (Received February 14, 1986; Accepted August 29, 1986)     

Ion Efflux during a Single Action Potential of Nitella axilliformis in Medium Lacking Ca2-

Ion efflux during excitation of Nitella axilliformis was measuredconductometrically. In medium lacking Ca²⁺ but with 0.1 mM MgCl₂,the duration of the action potential and the total efflux weremuch larger than those in APW, while the efflux rate, givenas the total efflux divided by the duration, was about halfof that in APW. (Received September 4, 1986; Accepted November 25, 1986)     

Intracellular Mobilization of Ca2+ and Inhibition of Cytoplasmic Streaming Induced by Transcellular Osmosis in Internodal Cells of Nitella flexilis

When transcellular osmosis was induced in internodal cells ofNitella flexilis that had been rendered inexcitable by treatmentwith KCl or EGTA, the rate of cytoplasmic streaming was reducedand the membrane was depolarized. In both KCl- and EGTA-treatedcells, the endoosmosis induced a significant increase in theconcentration of Ca²⁺ in the cytoplasm, which was demonstratedby monitoring the emission of light from aequorin that had beeninjected into the cytoplasm. When transcellular osmosis was induced in tonoplast-free cells,in which the intracellular Ca²⁺ concentration had been stabilizedat a very low level by treatment with the Ca²⁺-chelating agentEGTA, no change in the rate of cytoplasmic streaming on theendoosmosis side was observed. Hydration of the cytoplasm in the absence of endoosmosis wasinduced by direct introduction of a hypotonic medium into thevacuole by intracellular perfusion. The results mimicked theinhibition of streaming induced by transcellular osmosis. During transcellular osmosis, chloroplasts on the endoosmosisside swelled as a result of dilution of the cell sap. Swellingof chloroplasts was demonstrated to be unrelated to the inhibitionof streaming, since streaming was retarded at sites from whichchloroplasts had been removed. It is suggested that hydration of the cytoplasm triggers themobilization of Ca²⁺ from internal stores and causes an increasein the level of cytoplasmic Ca²⁺ that is responsible for theinhibition of streaming. Possible mechanisms for the osmosis-inducedincreases in the level of Ca²⁺ in the cytoplasm are discussed. (Received January 11, 1993; Accepted November 8, 1993)     

Identification of myosin in Nitella flexilis.

A myosin B-like protein was extracted from the alga Nitella flexilis. SDS-polyacrylamide gel electrophoresis revealed the presence of myosin heavy chain and actin as the main components. At high ionic strength, its ATPase [EC 3.6.1.3] reaction was activated by EDTA or Ca2+ and inhibited by Mg2+. At low ionic strength, superprecipitation was induced by the addition of ATP. Myosin was purified from Nitella myosin B. The molecular weight of the heavy chain of Nitella myosin, estimated by SDS-gel electrophoresis, was slightly higher than that of skeletal muscle myosin. At low ionic strength, Nitella myosin aggregated to form bipolar filaments about 0.2 micron long. At high ionic strength, its ATPase reaction was activated by EDTA or Ca2+, and inhibited by Mg2+. The Mg2+-ATPase reaction of Nitella myosin was activated by skeletal muscle F-actin.     

45Ca displacement related to pharmacologically induced prolonged action potentials in Nitella flexilis

Nitella cells were loaded with 45Ca2+ to an activity of 2 X 10(5) cpm. Insertion of two glass-capillary electrodes into each of six cells released varying amounts of Ca2+ in the order of 1 mumol per cell, but hyperpolarizing and depolarizing pulses up to 500 ms in duration caused no measurable loss (less than 57 pmol) of Ca2+ even when the latter elicited action potentials. Addition of 10 mumol of Ba2+ or tetraethylammonium (TEA) caused losses up to 1200 pmol of Ca2+ from the cells and prolonged the action potentials by a factor of three or more. Subsequent addition of Ba2+ or TEA to treated cells caused no further losses of Ca. Because prolonged action potentials can apparently only be elicited after the chelation or displacement of Ca2+, we propose that, as in many animal cells, the K+ channels responsible for the normal brief repolarizing phase of the action potential are controlled by Ca2+ in these electrically excitable plant cells.     

Ca2+ effect on protoplasmic streaming in Nitella internodal cell

Ca²⁺ ion effect on protoplasmic streaming in an internodal cell of Nitella has been investigated for various temperatures. We have found that the protoplasmic streaming at low temperature is remarkably affected by the Ca²⁺ ions in the internodal cell but larger concentrations of the Ca²⁺ ions are needed to suppress the streaming velocity at higher temperatures. These streaming behaviors of the protoplasm, furthermore, have been elucidated on the basis of the reaction equations which take into account ATP hydrolysis due to actin-myosin molecules and inactivity of the molecules due to the Ca²⁺ ions.     

Selectivity characteristics of cation channels in Nitella flexilis plasmalemma

Ionic selectivity of Nitella flexilis plasmalemma cation channels is studied by voltage-clamp method with consecutive replacing of cations in the bathing medium. The selectivity sequence received by measuring the ionic current reversal potentials, psi alpha is: Ba++ approximately equal to Sr++ approximately equal to Ca++ greater than Mg++ greater than Cs+ approximately equal to K+ greater than Na+ greater than Li+. An analysis of results based on the three-barrier channel model suggests that when ions of the same valency are compared, the channel selectivity is determined by specific interactions between the ion and the nearest water molecules, which is possible both in a narrow and wide pore. On the other hand, when monovalent and divalent ions are compared the effects of ions binding in the channel or near the membrane surface prevail, thus causing the channel preference for divalent cations.     

Temporal Relationship between Action Potential and Ca2+ Transient in Characean Cells

Temporal relationship between the action potential and the changein cytosolic Ca²⁺ concentration was investigated in cells offour species of Characeae, Chara corallina, Nitellopsis obtusa,Nitella flexilis and Nitella axilliformis. The Ca²⁺ transientwas detected by light emission from Ca²⁺-sensitive photoproteinaequorin injected into the cytoplasm. Action potential was triggeredby an outward or sometimes inward electric current pulse of20–50 ms in most cases. In all species the action potentialstarted at almost the same time as the time at which the lightemission from aequorin began to increase. Also the peak of actionpotential almost coincided with that of light emission, whichis in contrast with the slower Ca²⁺ transient in Chara reportedby Thiel et al. [(1997) J. Exp. Bot. 48: 609]. A discussionwas made on the origin of Ca²⁺ transient and the ionic processesduring membrane excitation. (Received July 2, 1998; Accepted October 5, 1998)     

Light-induced hyperpolarization of membrane potential in Nitella flexilis call plasmalemma

Light-induced hyperpolarization of the membrane potential in Nitella flexilis cell plasmalemma was investigated by the clamping method. It is shown that this response is of an excitation character. The equivalent electric diagram used in the work and the electromotive force included on its basis allowed consideration of two possible (from the common point of view) mechanisms of the response: the action of the electrogenic ion pump and membrane specific permeability to a definite ion. The membrane being in the hyperpolarization state is not capable of acting as a bicarbonate electrode. It is suggested that the response under study is defined by the action of the electrogenic pump, the role of which is performed by HCO2(3)-dependent H+-ATPase with the ion channel as an exciting one.     

Proton-Metal Cation Exchange in the Cell Wall of Nitella flexilis

When protons are exchanging for bivalent cations (Cu²⁺, Zn²⁺, or Ca²⁺) on the carboxylic groups of Nitella flexilis cell wall, the values of the respective global equilibrium constants do not change up to a protonation degree of 80%. These values drastically increase at higher proton concentrations and tend to 3.4, which is the intrinsic pK value of the constitutive α-d-galacturonic acid monomer. These data suggest that the electric field in the matricial polymer and the cation bridges between pairs of negative sites have disappeared.     

Temperature Sensitivity of the Tonoplast Ca2+-Activated K+ Channel in Chara: The Influence of Reversing the Sign of Membrane Potential

A detailed temperature dependence study of a well-defined plant ion channel, the Ca²⁺-activated K⁺ channel of Chara corallina, was performed over the temperature range of their habitats, 5–36°C, at 1°C resolution. The temperature dependence of the channel unitary conductance at 50 mV shows discontinuities at 15 and 30°C. These temperatures limit the range within which ion diffusion is characterized by the lowest activation energy (E _a = 8.0 ± 1.6 kJ/mol) as compared to the regions below 15°C and above 30°C. Upon reversing membrane voltage polarity from 50 to −50 mV the pattern of temperature dependence switched from discontinuous to linear with E _a = 13.6 ± 0.5 kJ/mol. The temperature dependence of the effective number of open channels at 50 mV showed a decrease with increasing temperature, with a local minimum at 28°C. The mean open time exhibited a similar behavior. Changing the sign of membrane potential from 50 to −50 mV abolished the minima in both temperature dependencies. These data are discussed in the light of higher order phase transitions of the Characean membrane lipids and corresponding change in the lipid-protein interaction, and their modulation by transmembrane voltage. Received: 14 June 2000/Revised: 20 September 2000     

Role of Ca2+ and Ca2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Causes of Abnormal Ca2+ Transients in Guinea Pig Pathophysiological Ventricular Muscle Revealed by Ca2+ and Action Potential Imaging at Cellular Level

Background

Abnormal Ca²⁺ transients are often observed in heart muscles under a variety of pathophysiological conditions including ventricular tachycardia. To clarify whether these abnormal Ca²⁺ transients can be attributed to abnormal action potential generation or abnormal Ca²⁺ handling/excitation-contraction (EC) coupling, we developed a procedure to determine Ca²⁺ and action potential signals at the cellular level in isolated heart tissues.

Methodology/Principal Findings

After loading ventricular papillary muscle with rhod-2 and di-4-ANEPPS, mono-wavelength fluorescence images from rhod-2 and ratiometric images of two wavelengths of emission from di-4-ANEPPS were sequentially obtained. To mimic the ventricular tachycardia, the ventricular muscles were field-stimulated in non-flowing Krebs solution which elicited abnormal Ca²⁺ transients. For the failed and alternating Ca²⁺ transient generation, there were two types of causes, i.e., failed or abnormal action potential generation and abnormal EC coupling. In cells showing delayed initiation of Ca²⁺ transients with field stimulation, action potential onset was delayed and the rate of rise was slower than in healthy cells. Similar delayed onset was also observed in the presence of heptanol, an inhibitor of gap junction channels but having a non-specific channel blocking effect. A Na⁺ channel blocker, on the other hand, reduced the rate of rise of the action potentials but did not result in desynchronization of the action potentials. The delayed onset of action potentials can be explained primarily by impaired gap junctions and partly by Na⁺ channel inactivation.

Conclusions/Significance

Our results indicate that there are multiple patterns for the causes of abnormal Ca²⁺ signals and that our methods are useful for investigating the physiology and pathophysiology of heart muscle.     

Ca2+及其信号转导途径与长时程增强的关系

Ca2 作为信号转导过程中的第二信使,参与机体的各种反应,尤其在神经突触可塑性方面,突触前后Ca2 浓度的变化发挥了重要的信息传递作用.Ca2 在长时程增强(long-term potentiation,LTP)过程中也发挥重要作用.它不仅是LTP产生的触发器,而且能通过激活下游的蛋白激酶、磷酸化ERK,以及活化即刻早期基因(IEGs)等促进基因转录和蛋白质合成,最终参与LTP的维持.