Effect of calcium ion on cytoplasmic streaming during turgor regulation in a brackish water charophyte Lamprothamnium

Abstract The brackish water charophyte Lamprothamnium succinctum regulates its turgor pressure against changes in the external osmotic pressure. Upon hypotonic treatment, the rate of cytoplasmic streaming in the internodal cells fell to almost zero, and then recovered to the original value within 20 min. The decrease could be inhibited by lowering the external Ca²⁺ concentration in the hypotonic medium. Also, cytoplasmic streaming in tonoplast-free cells of L. succintum was sensitive to Ca²⁺ like freshwater charophyte. Thus, the concentration of free Ca²⁺ in the cytoplasm seems to increase transiently upon hypotonic treatment.     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Vacuolar and cytoplasmic pH,ion composition,and turgor pressure in Lamprothamnium as a function of external pH

Ionic responses to alteration in external and internal pH were examined in an organism from a marine-like environment. Vacuolar pH (pH^v) is about 4.9–5.1, constant at external pH (pH^o) 5–8, while cytoplasmic pH (pH^c) increases from 7.3 to 7.7. pH^c regulation fails above pH^o 9, and this is accompanied by failure of turgor regulation. Na⁺ increases above pH^o 9, while K⁺ and Cl^– decrease. These changes alone cannot however explain the alterations in turgor. Agents known to affect internal pH are also tested for their effect on ion relations.Abbreviations C_i ion concentration - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - DES diethylstilbestrol - DMO 5,5-dimethyloxazolidine-2,4-dione - DNP 2,4-dinitrophenol - pH^o external pH - pH^c cytoplasmic pH - pH^v vacuolar pH - ⁱ osmotic pressure - turgor pressure     

Involvement of calcium ion in turgor regulation upon hypotonic treatment in Lamprothamnium succinctum

Abstract When internodal cells of Lamprothamnium succinetum were exposed to a hypotonic medium containing more than 1 mol m^?3 Ca²⁺, the elevated turgor pressure decreased and reached a steady state within 30–60 min. The hypotonic treatment caused the membrane potential to depolarize, with a time lag of ca. 1 min. The membrane conductance increased transiently with the same time lag and reached a peak value within 2–3 min. When the external Ca²⁺ concentration was lowered to 0.01 mol m^?3, both turgor regulation and change in the membrane conductance were strongly inhibited, although the membrane depolarization was not affected. When the Ca2+ level was returned to the normal level, the cells recovered their ability for turgor regulation and the membrane conductance attained a peak value after ca. 15–30 s. This response time is definitely shorter than that needed for the conductance change in cells exposed to a hypotonic medium having a normal level of Ca²⁺ from the beginning. We thus conclude that at least two sequential processes are involved in turgor regulation: a Ca²⁺ -independent process, followed by a Ca²⁺-dependent process.     

Net efflux of Cl− during hypotonic turgor regulation in a brackish water alga Lamprothamnium*

Abstract. Net efflux of Cl^? was measured potentiometrically (Ag/AgCl electrode) during turgor regulation which was induced by hypotonic treatment (hypotonic turgor regulation) in the alga Lamprothamnium succinctum. The efflux of Cl^? reached the peak value (11 μmol m ^?2s^?1) several minutes after the hypotonic treatment was started and then declined. The efflux of Cl^? and inhibition of the cytoplasmic streaming [reflection of an increase in cytoplasmic concentration of free Ca²⁺([Ca²⁺]_c)] were blocked under a low external concentration of Ca²⁺ ([Ca²⁺]_e) (0·01 mol m^?3) and resumed after raising [Ca²⁺]_e to the normal value (3·9 mol m^?3). The decrease in cell-osmotic pressure upon hypotonic treatment was inhibited by lowering either turgor pressure or [Ca^2h]_e. The inhibition was reflected in decreases of both the efflux of Cl^? and the membrane conductance. Recovery of the cytoplasmic streaming from the inhibition was also accelerated by the same treatments. It is concluded that an increase in turgor pressure is continuously sensed by the cells and that continuous presence of external Ca²⁺ is necessary for the hypotonic turgor regulation.     

Turgor regulation in a novel Halomonas species

The mechanisms by which a novel eubacterium, identified as belonging to the genus Halomonas, adapted to increases in the extracellular osmotic potential were investigated. It was shown that the ability of the bacterium to grow after hyperosmotic shock was dependent on the presence of potassium ions. Growth of the bacterium in 2 M NaCl medium could be limited by low concentrations of K⁺ and this enabled the affinity for K⁺ to be determined (K _s=21.5 M). Rubidium salts could be substituted for those of potassium, but the lowest concentration of Rb⁺ that allowed growth in 2 M NaCl medium was 50-fold greater than the minimum concentration of K⁺. ¹³C-NMR spectroscopy and HPLC analysis were used to demonstrate the accumulation of organic solutes in the cytoplasm after exposure to high salinities. The major osmolyte was ectoine, but glutamate and ectoine hydroxide were also present. Addition of exogenous glycine betaine to 3.25 M NaCl medium resulted in the accumulation of high intracellular concentrations of glycine betaine in the bacterium. This reduced the level of ectoine accumulation but did not fully inhibit the synthesis of this compound in the cytoplasm.Abbreviation Specific growth rate (generations/h)     

Turgor regulation inValonia macrophysa following acute osmotic shock

Summary The marine algaValonia macrophysa an inhabitant of shallow subtropical waters, is subjected to sudden dilutions of external seawater during rain showers. This study describes the mechanisms involved in turgor pressure regulation following acute hyposmotic shock. Turgor regulation is 88% effective and complete within 4 hr following hyposmotic shocks of up to –10 bar. Loss of vacuolar K⁺, Na⁺ and Cl^– accounts for the decrease in vacuolar osmotic pressure associated with turgor regulation. A novel mechanism of turgor regulation is exhibited byValonia macrophysa given hyposmotic shocks greater than about –4 bar. Such an osmotic shock causes cell wall tension to increase above a critical value of about 6×10⁵ dyne/cm, whereupon the protoplasm ruptures and the cell wall stretches irreversibly at a localized site. The protoplasm rupture is suggested by (1) a large abrupt increase in K⁺ efflux (as measured by⁸⁶Rb⁺), (2) a rapid decrease in turgor pressure as measured with a pressure probe, and (3) sudden depolarization of the vacuole potential. Evidence for an increase in cell wall permeability includes efflux from the vacuole of dextran (mol wt 70,000), which normally has a very low cell wall permeability, and scanning electron micrographs which show a trabeculated scar area in the cell wall. This mechanism of turgor regulation is physiologically important because 98% of the cells regained normal growth rate and turgor following acute osmotic shock.     

Increase in cytoplasmic calcium content in internodal cells of Lamprothamnium upon hypotonic treatment

Abstract Internodal cells of Lamprothamnium succinctum, a brackish water Characeae, regulate turgor pressure in response to changes in external osmotic pressure (turgor regulation). When internodal cells were transferred to a hypotonic medium containing 3.9 mol m^?3 Ca²⁺, the cell osmotic pressure decreased and the original turgor pressure was recovered. During turgor regulation Ca content of the cytoplasm increased significantly. Lowering the external Ca²⁺ concentration from 3.9 to 0.01 mol m^?3 inhibited this increase in cytoplasmic calcium content. In a hypotonic medium containing 0.01 mol m^?3 Ca²⁺, turgor regulation was inhibited as previously reported (Okazaki & Tazawa, 1986a). Thus transient increase in cytoplasmic Ca, probably in the ionized form, induced by hypotonic treatment may play an important role in turgor regulation.     

Turgor regulation in Lamprothamnium papulosum. I. I/V analysis and pharmacological dissection of the hypotonic effect

Current-voltage (I/V) analysis and pharmacological dissection were applied to membranes of Lamprothamnium at the time of hypotonic stress. At least three types of process were found to be involved in the response to this stress.

• 1 The first 10min of exposure to hypotonic medium resulted in a depolarization of about 50mV accompanied by a decrease or no change in conductance. This depolarization occurred with either K⁺ or Ca²⁺ (and consequently C^? channels inactivated.
• 2 The CI^? channels opened mainly in the first 15min of the hypotonic stress, increasing the membrane conductance by about an order of magnitude.
• 3 The K⁺ conductance rose as the Cl^? conductance started to diminish and reached a maximum after about 40 min.

Both types of channel were strongly potential-dependent with a conductance peak between -150 and 0mV. An inactivation of K⁺or CI^? channels resulted in moving the membrane potential away from the conductance maximum toward either E_K or E_CI, diminishing the ion efflux (and turgor regulation). The time courses of the conductance increases remained the same, suggesting that the conductance changes are not driven by feedback to some preset turgor level. The electrophysiology of the Lamprothamnium transporters is compared to that of salt-sensitive charophytes.     

The calmodulin-stimulated ATPase of maize coleoptiles is a 140000-Mr polypeptide

The calmodulin-stimulated ATPase of maize (Zea mays L.) coleoptiles has been purified by calcium-dependent binding to a calmodulin affinity column. In the presence of protease inhibitors (phenylmethylsulfonylfluoride and chymostatin) a polypeptide of relative molecular mass (M_r) 140000 (±10000) is obtained on sodium-dodecylsulphate polyacrylamide gels. This polypeptide is recognised specifically by an affinity-purified polyclonal antibody to mammalian calmodulin-stimulated calcium-pumping ATPases and is of similar M_r to the erythrocyte-membrane calcium pump (138000 M_r).Abbreviations EGTA ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - M_r apparent molecular mass - SDS sodium dodecyl sulphate     

Homeostatic regulation mechanism of spontaneous firing determines glutamate responsiveness in the midbrain dopamine neurons

Autonomous tonic firing of the midbrain dopamine neuron is essential for maintenance of ambient dopamine level in the brain, in which intracellular Ca²⁺ concentration ([Ca²⁺]c) plays a complex but pivotal role. However, little is known about Ca²⁺ signals by which dopamine neurons maintain an optimum spontaneous firing rate. In the midbrain dopamine neurons, we here show that spontaneous firing evoked [Ca²⁺]c changes in a phasic manner in the dendritic region but a tonic manner in the soma. Tonic levels of somatic [Ca²⁺]c strictly tallied with spontaneous firing rates. However, manipulatory raising or lowering of [Ca²⁺]c with caged compounds from the resting firing state proportionally suppressed or raised spontaneous firing rate, respectively, suggesting presence of the homeostatic regulation mechanism for spontaneous firing rate via tonic [Ca²⁺]c changes of the soma. More importantly, abolition of this homeostatic regulation mechanism significantly exaggerated the responses of tonic firings and high-frequency phasic discharges to glutamate. Therefore, we conclude that this Ca²⁺-dependent homeostatic regulation mechanism is responsible for not only maintaining optimum rate of spontaneous firing, but also proper responses to glutamate. Perturbation of this mechanism could cause dopamine neurons to be more vulnerable to glutamate and Ca²⁺ toxicities.     

Elucidation of the Mechanisms Underlying Hypo-osmotically Induced Turgor Pressure Regulation in the Marine Alga Valonia utricularis

Exposure of the giant marine alga Valonia utricularis to acute hypo-osmotic shocks induces a transient increase in turgor pressure and subsequent back-regulation. Separate recording of the electrical properties of tonoplast and plasmalemma together with turgor pressure was performed by using a vacuolar perfusion assembly. Hypo-osmotic turgor pressure regulation was inhibited by external addition of 300 microM of the membrane-permeable ion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In the presence of 100 microM NPPB, regulation could only be inhibited by simultaneous external addition of 200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a membrane-impermeable inhibitor of Cl(-) transport. At concentrations of about 100 microM, NPPB seems to selectively inhibit Cl(-) transporters in the tonoplast and K(+) transporters in the plasmalemma, whereas 300 microM NPPB inhibits K(+) and Cl(-) transporters in both membranes. Evidence was achieved by measuring the tonoplast and plasmalemma conductances (G(t) and G(p)) in low-Cl(-) and K(+)-free artificial seawater. Inhibition of turgor pressure regulation by 300 microM NPPB was accompanied by about 85% reduction of G(t) and G(p). Vacuolar addition of sulfate, an inhibitor of tonoplast Cl(-) transporters, together with external addition of DIDS and Ba(2+) (an inhibitor of K(+) transporters) also strongly reduced G(p) and G(t) but did not affect hypo-osmotic turgor pressure regulation. These and many other findings suggest that KCl efflux partly occurs via electrically silent transport systems. Candidates are vacuolar entities that are disconnected from the huge and many-folded central vacuole or that become disconnected upon disproportionate swelling of originally interconnected vacuolar entities upon acute hypo-osmotic challenge.     

Relation between cytoskeleton,hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells

Summary Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca²⁺ or Ca²⁺-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca²⁺ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K⁺ ionophore valinomycin.     

Specific inhibitors of intracellular Ca2+ transport ATPases

Support from the National Institutes of Health and the American Heart Association is gratefully acknowledged.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin     

Renal handling of Ca2+ in diabetes

Ca²⁺ transport in kidney has gained considerable attention in the recent past. Our laboratory has been involved in understanding the regulatory mechanisms underlying Ca²⁺ transport in the kidney across the renal basolateral membrane. We have shown that ANP, a cardiac hormone, mediates its biological functions by acting on its receptors in the kidney basolateral membrane. Furthermore, it has been established that ANP receptors are coupled with Ca²⁺ ATPase, the enzyme that participates in the vectorial translocation of Ca²⁺ from the tubular lumen to the plasma. It is possible that a defect in the ANP-receptor-effector system in diabetes (under certain conditions such as hypertension) may be associated with abnormal Ca²⁺ homeostasis and the development of nephropathy. Accordingly, future studies are needed to establish this hypothesis.     

Inhibition by Orthovanadate of ATP-Dependent Ca2+ Transport in Microsomes Isolated from Rat Liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5′-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca²⁺ transport activity (half-maximal inhibition at ～10 μM vanadate, corresponding to ～12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca²⁺ transport is noncompetitive with respect to added Ca²⁺ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca²⁺ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.     

Is the Ca2+-ATPase from sarcoplasmic reticulum also a heat pump?

We calculate, using the first law of thermodynamics, the membrane heat fluxes during active transport of Ca²⁺ in the Ca²⁺-ATPase in leaky and intact vesicles, during ATP hydrolysis or synthesis conditions. The results show that the vesicle interior may cool down during hydrolysis and Ca²⁺-uptake, and heat up during ATP synthesis and Ca²⁺-efflux. The heat flux varies with the SERCA isoform. Electroneutral processes and rapid equilibration of water were assumed. The results are consistent with the second law of thermodynamics for the overall processes. The expression for the heat flux and experimental data, show that important contributions come from the enthalpy of hydrolysis for the medium in question, and from proton transport between the vesicle interior and exterior. The analysis give quantitative support to earlier proposals that certain, but not all, Ca²⁺-ATPases, not only act as Ca²⁺-pumps, but also as heat pumps. It can thus help explain why SERCA 1 type enzymes dominate in tissues where thermal regulation is important, while SERCA 2 type enzymes, with their lower activity and better ability to use the energy from the reaction to pump ions, dominate in tissues where this is not an issue.

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.