Phosphatidylserine synthesis in rat cerebral cortex: effects of hypoxia,hypocapnia and development

Phosphatidylserine, which is necessary for protein kinase C activity, is synthesized in mammalian tissues by the Ca²⁺-dependent base exchange enzyme. The synthesis of phosphatidylserine is greater in slices or homogenates of rat cerebral cortex subjected to hypoxia by N₂ treatment when compared with O₂ plus 5% CO₂. An intermediate effect was observed when the treatment was done with N₂ plus 5% CO₂. Incorporation rates were dependent on Ca²⁺ in Krebs-Henseleit Ringer bicarbonate medium, being greater with 2 mM Ca²⁺ than with the same medium prepared without Ca²⁺. The increase of phosphatidylserine synthesis, due to hypoxia, was, on the contrary, more evident in the medium lacking added Ca²⁺. Similar results were obtained with the homogenates. This suggests that elevation of intracellular Ca²⁺, caused by hypocapnia and hypoxia, may be responsible for the greater incorporation of serine into phosphatidylserine. In both cerebrocortical slices and homogenate, [¹⁴C]serine incorporation decreased with development both in O₂ plus 5% CO₂ and N₂-treated preparations. However, in younger rats (14–18 days) hypoxia induced a lesser increase of phosphatidylserine than in 40 day old animals. We suggest that a regulatory mechanisms for phosphatidylserine synthesis is established during development and that N₂-treatment can increase phosphatidylserine synthesis by interfering with this regulatory mechanism.Abbreviations KRB Krebs-Henseleit Ringer bicarbonate - KRP Krebs Ringer phosphate - PS serine glycerophospholipids     

Effects of different types of acidosis and Ca2+ on cardiac contractility in the flounder (Pleuronectes flesus)

Summary In contrast to most other fish the isolated myocardium of flounder (Pleuronectes flesus) was able to maintain contractility well under acidosis. The response to acidosis was biphasic, the initial force decline being followed by spontaneous recovery under ongoing acidification. This reaction took place only under hypercapnia and did not appear when extracellular pH was decreased to the same extent by lowering bicarbonate. Biphasic response to hypercapnia was more pronounced with low than with high extracellular Ca²⁺. A depressed sensitivity towards extracellular Ca²⁺ was seen in hypercapnia in contrast to acidification by lowering bicarbonate. This indicates that hypercapnia in inducing severe intracellular acidification, triggers an efficient compensatory reaction which is modified by Ca²⁺-availability.     

Control of Ca2+ efflux and cyclic AMP by 5-hydroxytryptamine and dopamine in abalone gill.

5-Hydroxytrptamine increased the rate of Ca²⁺ efflux and the concentration of endogenous cyclic AMP in abalone gill in both 10 mM and 50 mM CaCl₂ concentrations externally. Dopamine decreased the rate of Ca²⁺ efflux in 50 mM CaCl₂ but slightly increased the efflux rate in 10 mM CaCl₂. At both external Ca²⁺ concentrations, dopamine increased the endogenous cyclic AMP concentration in the gill. 5-Hydroxytryptamine but not dopamine was found to activate adenylate cyclase in broken cell preparations of abalone gill. Cyclic AMP-dependent protein kinase activity was also demonstrated in homogenate fractions of abalone gill. It is suggested that both Ca²⁺ and cyclic AMP act as second messengers in the response of abalone gill to 5-hydroxytryptamine and dopamine.     

Calcium Effects on Stomatal Movement in Commelina communis L. : Use of EGTA to Modulate Stomatal Response to Light, KCl and CO(2)

Stomatal movements depend on both ion influx and efflux; attainment of steady state apertures reflects modulation of either or both processes. The role of Ca²⁺ in those two processes was investigated in isolated epidermal strips of Commelina communis, using the Ca²⁺ chelator EGTA to reduce apoplastic [Ca²⁺]. The results suggest that a certain concentration of Ca²⁺ is an absolute requirement for salt efflux and stomatal closure. EGTA (2 millimolar) increased KCl-dependent stomatal opening in darkness and completely inhibited the dark-induced closure of initially open stomata. Closure was inhibited even in a KCl-free medium. Thus, maintenance of stomata in the open state does not necessarily depend on continued K⁺ influx but on the inhibition of salt efflux. Opening in the dark was stimulated by IAA in a concentration-dependent manner, up to 15.4 micrometer without reaching saturation, while the response to EGTA leveled off at 9.2 micrometer. IAA did not inhibit stomatal closure to the extent it stimulated opening. The response to IAA is thus consistent with a primary stimulation of opening, while EGTA can be considered a specific inhibitor of stomatal closing since it inhibits closure to a much larger degree than it stimulates opening. CO₂ causes concentration-dependent reduction in the steady state stomatal aperture. EGTA completely reversed CO₂-induced closing of open stomata but only partially prevented the inhibition of opening.     

Internal transport of CO2 from the root‐zone to plant shoot is pH dependent

We investigated the fate of carbon dioxide (CO₂) absorbed by roots or internally produced by respiration using gas exchange and stable isotopic labeling. CO₂ efflux from detached leaves supplied with bicarbonate/CO₂ solutions was followed over six cycles. CO₂ effluxes were detected when bicarbonate solution at high pH was used, corresponding to 71–85% of the expected efflux. No CO₂ efflux was detected when CO₂ solutions at low pH were used but CO₂ efflux was subsequently detected as soon as bicarbonate solutions at high pH were supplied. By sealing the leaf and petiole in a plastic bag to reduce diffusion to the atmosphere, a small CO₂ efflux signal (14–30% of the expected efflux) was detected suggesting that CO₂ in the xylem stream can readily escape to the atmosphere before reaching the leaf. When the root‐zones of intact plants were exposed to CO₂ solutions, a significant efflux from leaf surface was observed (13% of the expected efflux). However, no signal was detected when roots were exposed to a high pH bicarbonate solution. Isotopic tracer experiments confirmed that CO₂ supplied to the root‐zone was transported through the plant and was readily lost to the atmosphere. However, little ¹³C moved to the shoot when roots were exposed to bicarbonate solutions at pH 8, suggesting that bicarbonate does not pass into the xylem.     

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells

The removal of extracellular HCO⁻₃ together with a decrease in pCO₂, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of ⁴⁵Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca²⁺ and abolished at low extracellular Na⁺ concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca²⁺. The removal of HCO⁻₃ and decrease in the pCO₂ also reduced the magnitude of both the secondary rise in ⁴⁵Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca²⁺ from islet cells as mediated by Na⁺-Ca²⁺ countertransport and the inflow of Ca²⁺ by gated Ca²⁺ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.     

Comparative study of Y<Superscript>3+</Superscript> effect on calcium-dependent processes in frog cardiac muscle and mitochondria of rat cardiomyocytes

Inotropic effects of yttrium acetate (Y³⁺) on contractions of myocardium preparations of the frog Rana ridibunda, as well as on respiration and the inner membrane potential (ΔΨ_mito) of isolated rat heart mitochondria were studied. 2 mM yttrium in Ringer solution was found to significantly reduce the amplitude of myocardium contractions, evoked by electric stimulation, and increase the half-relaxation time (n = 5). In experiments with Ca²⁺, Y³⁺ decreased the Ca²⁺-dependent basal respiration rate in rat heart mitochondria, energized by glutamate and malate, impeded the reduction in respiration of these mitochondria operating in state 3 after Chance or uncoupled by 2,4-dinitrophenol, and inhibited a Ca²⁺-induced reduction in their inner membrane potential. The data obtained are important for better understanding the mechanism underlying Y³⁺ effects on the myocardial Ca²⁺-dependent processes. Possible mechanisms of the negative inotropic effect of Y³⁺ on myocardium and its influence on the Ca²⁺-dependent processes in rat mitochondria are discussed.     

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

Changes in free-calcium levels and pH in synaptosomes during transmitter release

The free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in rat brain synaptosomes estimated by the indicator quin 2 is 104±8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca²⁺), but decreases at lower Ca²⁺ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (γ-aminobutyric acid) or the release induced by K⁺ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K⁺ causes an abrupt increase in [Ca]_i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]_i. The increase in [Ca]_i produced by K⁺ depolarisation does not occur in the absence of Ca²⁺ in the medium. The data are consistent with a direct correlation between [Ca_i] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04±0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K⁺ depolarisation.     

Alkaline pH and Internal Calcium Increase Na+ and K+ Effluxes in LK Sheep Red Blood Cells in Cl−-free Solutions

We examined the effects of pH, internal ionized Ca (Ca²⁺ _i ), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K⁺ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K⁺ phenotypes. In LK SRBCs the K⁺ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K⁺ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K⁺ efflux was activated (100%) when Ca²⁺ _i was increased (+A23187, +Ca²⁺ _o ) into the μm range. However, in comparison to human red blood cells, the Ca²⁺ _i -induced increase in K⁺ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na⁺ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca²⁺ _i . In contrast, in HK SRBCs the K⁺ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca²⁺ _i . These studies suggest the presence in LK SRBCs, of at least two pathways for Cl⁻-independent K⁺ and Na⁺ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca²⁺ _i . Received: 23 May 1996/Revised: 6 December 1996     

Rapid calcium exchange for protons and potassium in cell walls of Chara

Net fluxes of Ca²⁺, H⁺ and K⁺ were measured from intact Chara australis cells and from isolated cell walls, using ion-selective microelectrodes. In both systems, a stimulation in Ca²⁺ efflux (up to 100 nmol m^?2 s^?1, from an influx of ～40 nmol m^?2 s^?1) was detected as the H⁺ or K⁺ concentration was progressively increased in the bathing solution (pH 7.0 to 4.6 or K⁺ 0.2 to 10mol m^?3, respectively). A Ca²⁺ influx of similar size occurred following the reverse changes. These fluxes decayed exponentially with a time constant of about 10 min. The threshold pH for Ca²⁺ efflux (pH 5.2) is similar to a reported pH threshold for acid-induced wall extensibility in a closely related characean species. Application of NH₄⁺ to intact cells caused prolonged H⁺ efflux and also transient Ca²⁺ efflux. We attribute all these net Ca²⁺ fluxes to exchange in the wall with H⁺ or K⁺. A theoretical treatment of the cell wall ion exchanges, using the ‘weak acid Donnan Manning’ (WADM) model, is given and it agrees well with the data. The role of Ca²⁺ in the cell wall and the effect of Ca²⁺ exchanges on the measured fluxes of other ions, including bathing medium acidification by H⁺ efflux, are discussed.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Magnesium–calcium signalling in rat parotid acinar cells: effects of acetylcholine

This study investigated the effects of extracellular Mg²⁺ ([Mg²⁺]_o) on basal and acetylcholine (ACh)-evoked amylase secretion and intracellular free Ca²⁺ ([Ca²⁺]_i) in rat parotid acinar cells. In a medium containing 1.1 mM [Mg²⁺]_o, ACh evoked significant increases in amylase secretion and [Ca²⁺]_i. Either low (0 mM) or elevated (5 and 10 mM) [Mg²⁺]_o attenuated ACh-evoked responses. In a nominally Ca²⁺ free medium, elevated [Mg²⁺]_o attenuated basal and ACh-evoked amylase secretion and [Ca²⁺]_i. In parotid acinar cells incubated with either 0, 1.1, 5 or 10 mM [Mg²⁺]_o, ACh evoked a gradual decrease in [Mg²⁺]_i. These results indicate that the ACh-evoked Mg²⁺ efflux is an active process since Mg²⁺ has to move against its gradient. Either lidocaine, amiloride, N-methyl-d-glucamine, quinidine, dinitrophenol or bumetanide can elevate [Mg²⁺]_i above basal level. In the presence of these membrane transport inhibitors, ACh still evoked a decrease in [Mg²⁺]_i but the response was less pronounced with either [Na⁺]_o removal or in the presence of either amiloride or quinidine. These results indicate marked interactions between Ca²⁺ and Mg²⁺ signalling in parotid acinar cells and that ACh-evoked Mg²⁺ transport was not dependent upon [Na⁺]_o.     

Systemic signalling in barley through action potentials

Using apoplastic voltage- and ion selective microprobes, in barley leaves action potentials (APs) have been measured, which propagate acropetally as well as basipetally from leaf to leaf or from root to leaf following the application of mild salt stress (e.g. 30–50 mM KCl or NH₄Cl) or amino acids (e.g. 1 mM glutamic acid or 5 mM GABA). Voltage changes were biphasic, followed an ‘all-or-none’ characteristic, and propagated at 20–30 cm min⁻¹ irrespective of the direction. With the salt-induced APs, a strong initial depolarization is the main AP-releasing factor that first causes Ca²⁺ influx and then anion efflux. Ca²⁺ influx coincides with an initial slower depolarization, the rapid anion efflux causes the typical voltage ‘break-through’. Subsequently, K⁺-efflux starts after the depolarizing voltage has passed the K⁺ equilibrium potential (inversion of the K⁺ driving force). Glutamic acid and GABA induce APs not through membrane depolarization, but presumably by binding to a putative receptor or to ligand-gated Ca²⁺-conducting channels, respectively, followed by Ca²⁺ induced activation of anion efflux. APs are accompanied by transient apoplastic pH increase (about 1 unit), and by cytoplasmic pH decrease (about 0.5 units). The apoplastic pH change is interpreted as an indicator of stress, the cytoplasmic pH change as a prerequisite for defence related gene activation. Since APs are released by agents added in a moderate concentration range, it is suggested that they may serve as first and fast systemic signals following attack from pathogens.     

Energy metabolism and intracellular pH in trout heart muscle under anoxia and different [Ca2+]0

Summary Electrically stimulated, isometrically contracting heart ventricular strips of rainbow trout were subjected to 60 min of anoxia. During this period the twitch force fell by about 75% and the production of lactate increased. The tissue concentration of ATP remained unchanged, whereas a considerable drop in creatine phosphate (CP) occurred. The intracellular pH (pH_i), as estimated with the DMO-method, did not change significantly. In some experiments the extracellular calcium concentration, [Ca²⁺]₀, was increased and maintained at 5 instead of 1.25 mM for the last 30 min of anoxia. At this concentration the force developed was about twice the level observed at low [Ca²⁺]₀ and lactate production was further enhanced. Other parameters were not significantly affected by the increase in [Ca²⁺]₀. It is suggested that the excitation-contraction coupling is a target of impairment in the anoxic trout heart.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

Preload-induced changes in isometric tension and [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in rat myocardium

Preload-induced changes of active tension and [Ca²⁺]i are “dissociated” in mammalian myocardium. This study aimed to describe the distinct effects of preload at low and physiological [Ca²⁺]_o. Rat RV papillary muscles were studied in isometric conditions at 25‡C and 0.33 Hz at 1 mM (hypo-Ca group) and 2.5 mM [Ca²⁺]_o (normal-Ca group). [Ca²⁺]_i was monitored with fura-2/AM. Increase of preload caused a rise of active tension in hypo-Ca and normal-Ca groups whereas peak fluorescence rose significantly only at low [Ca²⁺]_o. End-diastolic tension, end-diastolic level of fluorescence, time-to-peak tension, but not time-to-peak of Ca²⁺ transient, progressively increased with preload. Mechanical relaxation decelerated with preload while Ca²⁺ transient decay time decreased in the initial phase and increased in the late phase, resulting in a prominent “bump” configuration. The “bump” was assessed as a ratio of its area to the fluorescence trace area. It was a new finding that the preload-induced rise of this ratio was twice as large in hypo-Ca. Our results indicate that preload-induced changes in active tension and [Ca²⁺]_i are “dissociated” in rat myocardium, with relatively higher expression at low [Ca²⁺]_o. Ca-dependence of Ca-TnC association/dissociation kinetics is thought to be a main contributor to these preload-induced effects.     

Effects of adenosine diphosphate on Ca fluxes and Ca accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Glutamate-supported calcium movements in rat liver mitochondria effects of anions and pH

Ca²⁺ efflux from rat liver mitochondria in the presence of glutamate is stimulated by a decrease in pH from 7.3 to 6.8 and the rate is dependent on the phosphate concentration. During Ca²⁺ (13 μm) uptake and release at low pH (+ phosphate), swelling is minimal, but a large oxidation of pyridine nucleotides and sustained membrane depolarization occurs. The depolarization (but not Ca²⁺ efflux) is reversed by ruthenium red. An absolute requirement for phosphate to support Ca²⁺ efflux is demonstrated by using acetate or lactate to support Ca²⁺ uptake (efflux is depressed at pH 6.8). Preincubation with mersalyl, to block phosphate movements, with subsequent phosphate addition preceeding Ca²⁺ uptake also inhibits efflux. β-Mercaptoethanol then stimulates efflux concomittent with membrane repolarization. Ca²⁺ efflux is not a simple result of collapse of ΔpH since nigericin inhibits phosphate transport and Ca²⁺ release. Following Ca²⁺ uptake at pH 6.8, respiratory inhibition occurs, but oxygen consumption coupled to ATP synthesis can be stimulated by succinate (+ rotenone). Addition of succinate allows reuptake of Ca²⁺, reduction of pyridine nucleotides, and repolarization of the membrane potential. Respiratory inhibition is also seen with nigericin, but no Ca²⁺ efflux is observed. Coupled respiration with glutamate is seen at pH 6.8 following Ca²⁺ uptake in the presence of lactate with subsequent addition of phosphate to promote Ca²⁺ efflux. We conclude that Ca²⁺ efflux is not a consequence of respiratory inhibition, but is mediated solely by phosphate movements. The inhibitory effect of Mg²⁺ on Ca²⁺ efflux is probably due to Mg²⁺-dependent inhibition of the Ca²⁺ diffusion potential so that the compensatory increase in ΔpH due to membrane depolarization does not occur and phosphate entry is slowed.     

NO MECHANISTIC DEPENDENCE OF PHOTOSYNTHESIS ON CALCIFICATION IN THE COCCOLITHOPHORID EMILIANIA HUXLEYI (HAPTOPHYTA)1

There is still considerable uncertainty about the relationship between calcification and photosynthesis. It has been suggested that since calcification in coccolithophorids is an intracellular process that releases CO₂, it enhances photosynthesis in a manner analogous to a carbon‐concentrating mechanism (CCM). The ubiquitous, bloom‐forming, and numerically abundant coccolithophorid Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler was studied in nutrient‐replete, pH and [CO₂] controlled, continuous cultures (turbidostats) under a range of [Ca²⁺] from 0 to 9 mM. We examined the long‐term, fully acclimated photosynthesis‐light responses and analyzed the crystalline structure of the coccoliths using SEM. The E. huxleyi cells completely lost their coccosphere when grown in 0 [Ca²⁺], while thin, undercalcified and brittle coccoliths were evident at 1 mM [Ca²⁺]. Coccoliths showed increasing levels of calcification with increasing [Ca²⁺]. More robust coccoliths were noted, with no discernable differences in coccolith morphology when the cells were grown in either 5 or 9 mM (ambient seawater) [Ca²⁺]. In contrast to calcification, photosynthesis was not affected by the [Ca²⁺] in the media. Cells showed no correlation of their light‐dependent O₂ evolution with [Ca²⁺], and in all [Ca²⁺]‐containing turbidostats, there were no significant differences in growth rate. The results show unequivocally that as a process, photosynthesis in E. huxleyi is mechanistically independent from calcification.