Magnesium homeostasis in cardiac cells

Several aspects of Mg²⁺ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg²⁺ indicator, FURAPTRA. The concentration of cytosolic Mg²⁺ ([Mg²⁺]_i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg²⁺]_i below electrochemical equilibrium, we manipulated the Na⁺ gradient and assessed the effects on [Mg²⁺]_i. When extracellular Na⁺ was removed, [Mg²⁺]_i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg²⁺]_i, which was dependent upon extracellular Ca²⁺, was observed when intracellular Na⁺ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca²⁺ can modulate [Mg²⁺]_i. In addition, removing extracellular Na⁺ caused a decrease in intracellular pH (pH_i), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca²⁺ and H⁺ interact intracellularly. Since changes in the Na⁺ gradient can also alter pH_i, we questioned whether pH can modulate [Mg²⁺]_i. pH_i was manipulated by the NH₄Cl prepulse method. NH₄ ⁺-evoked changes in pH_i, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg²⁺]_i; [Mg²⁺]_i changed by –0.16 mM/unit pH. These NH₄ ⁺-evoked changes in [Mg²⁺]_i were not caused by movements of Mg₂₊ or Ca²⁺ across the sarcolemma or by changes in cytosolic Ca²⁺. Additionally, pH_i was manipulated by changing extracellular pH (pH_o). When pH_o was decreased from 7.4 to 6.3, pH_i decreased by 0.64 units and [Mg₂₊]_i increased by 0.12 mM; in contrast, when pH_o was raised from 7.4 to 8.3, pH_i increased by 0.6 units and [Mg²⁺]_i did not change significantly. The results of our investigations suggest that Ca ²⁺ and H⁺ can modulate [Mg²⁺]_i, probably by affecting cytosolic Mg²⁺ binding and/or subcellular Mg²⁺ transport and that such redistribution of intracellular Mg²⁺ may play an important role in Mg²⁺ homeostasis in cardiac cells.     

Importance of sodium ion to the progesterone-initiated acrosome reaction in human sperm

Progesterone (P) has previously been shown to rapidly increase free intracellular calcium concentration ([Ca²⁻]_i), and subsequently to initiate the acrosome reaction (AR) in capacitated human sperm. The present study used cytochemical analysis of the AR, and spectrofluorometric determination of sperm [Ca²⁻]_i and intracellular pH (pH_i) in Na⁺-containing and Na⁺-deficient bicarbonate/CO₂-buffered media to investigate the role of Na⁺ in these P-initiated changes. We found that P failed to initiate the AR in Na⁺-deficient medium, and that the initial rise in [Ca²⁺]_i following P (1 μg/ml) stimulation was similar for both media; however, the [Ca²⁺]_i in the Na⁺-deficient medium regressed more rapidly and plateaued at a significantly lower [Ca²⁺]_i. Moreover, the differences in plateau [Ca²⁺]_i were directly related to the percentage of acrosome reactions, suggesting that the plateau phase is not due to [Ca²⁺]_i, but rather to the release of intracellular fura-2 into the medium during the AR. These [Ca²⁺]_i and AR results are in contrast to those reported previously by others for human sperm and suggest that a Na⁺-dependent mechanism is important in the P-initiated human sperm AR. Such a Na⁺ requirement may reflect the involvement of this ion in pH_i regulation, as capacitated sperm that were incubated in a Na⁺-deficient medium for ≥ 30 min displayed a significantly lower pH_i. © 1996 Wiley-Liss, Inc.     

Extracellular ATP Stimulates Calcium Influx in Neuroblastoma Glioma Hybrid NG108–15 Cells

Abstract— ATP-induced changes in the intracellular Ca²⁺concentration ([Ca²⁺]_i) in neuroblastoma glioma hybrid NG108–15 cells were studied. Using the fluorescent Ca²⁺indicator fura-2, we have shown that the [Ca²⁺]_i increased in response to ATP. ATP at 3 mM caused the greatest increase in [Ca^z+]_i, whereas at higher concentrations of ATP the response became smaller. Two nonhydrolyzable ATP analogues, adenosine 5′-thiotriphosphate and 5′-adenylyl-β, γ-imidodiphosphate, could not trigger significant [Ca²⁺]_i change, but they could block the ATP effect. Other adenine nucleotides, including ADP, AMP, α,β-methylene-ATP, β,γ-methylene-ATP, and 2-methylthio-ATP, as well as UTP and adenosine, all had no effect on [Ca²⁺]_i at 3 mM. In the absence of extracellular Ca²⁺, the effect of ATP was inhibited totally, but could be restored by the addition of Ca²⁺ to the cells. Upon removal of Mg²⁺, the maximum increase in [Ca²⁺]_i induced by ATP was enhanced by about 42%. Ca²⁺-channel blockers partially inhibited the ATP-induced [Ca²⁺]_i rise. The ATP-induced [Ca²⁺]_i rise was not affected by thapsigargin pretreatment, though such pretreatment blocked bradykinin-induced [Ca²⁺]_i rise completely. No heterologous desensitization of [Ca²⁺]_i rise was observed between ATP and bradykinin. The magnitude of the [Ca²⁺]_i rise induced by ATP increased between 1.5 and 3.1 times when external Na⁺was replaced with Tris, N-methyl-d -glucamine, choline, or Li⁺. The addition of EGTA or verapamil to cells after their maximum response to ATP immediately lowered the [Ca²⁺]_i to the basal level in Na⁺-containing or Na⁺-free Tris solution. Our results suggest that ATP stimulates Ca²⁺influx via at least two pathways: ion channels that are permeable to Ca²⁺ and Na⁺, and pores formed by ATP^4-.     

Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics

Cultured cerebellar granule cells were co-loaded with Ca²⁺-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (????_m). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca²⁺]ⁱ. This sMD was insensitive to a removal of external Ca²⁺ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca²⁺ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6?C8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]_c) and ????_m changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]_c and ????_m. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ????_m despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca²⁺ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.     

Effects of External pH Variations on Brain Presynaptic Sodium and Calcium Channels; Repercussion on the Evoked Release of Amino Acid Neurotransmitters

The effects of external pH (pH _out) variations on the Na⁺ and on the Ca²⁺ dependent fractions of the evoked amino acid neurotransmitter release were separately investigated, using GABA as a model transmitter. In [³H]GABA loaded mouse brain synaptosomes, the external acidification (pH _out6.0) markedly decreased the Na⁺ dependent fraction of [³H]GABA release evoked by veratridine (10 M) in the absence of external Ca²⁺, as well as the Ca²⁺ dependent fraction of [³H]GABA release evoked by high (20 mM) K⁺ in the absence of external Na⁺. The depolarization-induced elevation of [Na _i ] (monitored in synaptosomes loaded with the Na⁺ indicator dye, SBFI) and the depolarization-induced elevation of [Ca _i ] (monitored in synaptosomes loaded with the Ca²⁺ indicator dye fura-2) were also markedly decreased at pH _out 6. On the contrary, the external alkalinization (pH _out 8) facilitated all the above responses. A slight increase of the baseline release of the [³H]GABA was observed when pH _out was changed from 7.4 to 8. This effect was only observed in the presence of Ca²⁺. pH _out changes from 7.4 to 6 or to 7 did not modify the baseline release of the transmitter. All the effects of pH _out variations on [³H]GABA release were independent on the presence of HCO-₃. It is concluded that external H⁺ regulate amino acid neurotransmitter release by their actions on presynaptic Na⁺ channels, as well as on presynaptic Ca²⁺ channels.     

A decrease of both [Ca]e and [H]e produces cell damage in the perfused rat heart

Cell damage of the Langendorff-perfused rat heart in response to a decrease of both [Ca²⁺]_e and [H⁺]_e is described. At pH_e = 7.7, lactate dehydrogenase (LDH) release could be induced during perfusion with media of reduced [Ca²⁺]_e (0.1–0.4 mmol/I). Decreasing pH_e to normal abolished LDH release. The gap junction channel blocker heptanol (2 mmol/I) also reduced enzyme release, and polyethylene glycol (9% PEG₆₀₀₀) totally prevented cell damage. Elevation of buffer capacity of perfusion media or perfusion flow both increased LDH release. Cell damage could also be aggravated by substituting 10 mmol/I of [Na⁺]_e by foreign cations. At [Ca²⁺]_e = 0.1 mmol/I and pH_e = 7.7, [Ca²⁺]_i and [Na⁺]_i of non-lysed cells were markedly increased (in HCO₃/CO₂ buffered media to about 7.0 μmol/I and 36 mmol/I, respectively; in HEPES-buffered media, to about 5.0 μmol/I and 55 mmol/l; physiological values of [Ca²⁺]_i and [Na⁺]_i are around 0.1 μmol/I and 10 mmol/I, respectively), whereas pH_i was not appreciably elevated. In contrast to myocytes in the intact heart, [Ca²⁺]_i of isolated cardiomyocytes under similar conditions was decreased to about 75 nmol/I and LDH release was negligible; pH_i of isolated cardiomyocytes, as in intact myocardium, did not change appreciably. The results indicate that Ca²⁺ overload is produced at lowered [Ca²⁺]_e and [H⁺]_e by an influx of Ca²⁺ through gap junctional leaks.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Acceleration of Sodium-Calcium Exchange Activity during ATP-induced Calcium Release in Transfected Chinese Hamster Ovary Cells

The P_2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca²⁺]_i due to Ca²⁺ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca²⁺ release was accompanied by a Ca²⁺- dependent regulatory activation of Na⁺/Ca²⁺ exchange activity: Addition of extracellular Ca²⁺ (0.7 mM) 0–1 min after ATP evoked a dramatic rise in [Ca²⁺]_i in Na⁺-free media (Li⁺ substitution) compared to Na⁺-containing media; no differences between Na⁺- and Li⁺-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na⁺ and Ca²⁺, the ATP-evoked rise in [Ca²⁺]_i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca²⁺]_i which eventually came to exceed the declining [Ca²⁺]_i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba²⁺ influx, consistent with regulatory activation of the Na⁺/Ca²⁺ exchanger. The acceleration of Ba²⁺ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na⁺ or when the Ca²⁺ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca²⁺]_i under Na⁺-free conditions, but did not inhibit the ATP-evoked stimulation of Ba²⁺ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca²⁺ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca²⁺ release from intracellular stores through regulatory activation by increased [Ca²⁺]_i. In the absence of extracellular Ca²⁺, the stimulation of exchange activity is short-lived and follows the time course of the [Ca²⁺]_i transient; in the presence of extracellular Ca²⁺, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca²⁺ entry.     

Hypoxia‐induced increase in intracellular calcium concentration in endothelial cells: Role of the Na+‐glucose cotransporter

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet‐activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca²⁺]_i) which then activates the phospholipase A₂ (PLA₂). The link between the decrease in ATP and the increase in [Ca²⁺]_i was not known and is investigated in this work. We first showed that the presence of extracellular Na⁺ was necessary to observe the hypoxia‐induced increase in [Ca²⁺]_i and the activation of PLA₂. This increase was not due to the release of Ca²⁺ from intracellular stores, since thapsigargin did not inhibit this process. The Na⁺/Ca²⁺ exchanger was involved since dichlorobenzamil inhibited the [Ca²⁺]_i and the PLA₂ activation. The glycolysis was activated, but the intracellular pH (pH_i) in hypoxic cells did not differ from control cells. Finally, the hypoxia‐induced increase in [Ca²⁺]_i and PLA₂ activation were inhibited by phlorizin, an inhibitor of the Na⁺‐glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na⁺ through the activated Na⁺‐glucose cotransport followed by the activation of the Na⁺/Ca²⁺ exchanger, resulting in a net influx of Ca²⁺. J. Cell. Biochem. 84: 115–131, 2002. © 2001 Wiley‐Liss, Inc.     

Intracellular free sodium concentrations in GH4C1 cells

In the present investigation, intracellular sodium ([Na⁺]_i) levels were determined in GH₄C₁ cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na⁺]_i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na⁺. Resting [Na⁺]_i in GH₄C₁ cells was 26 ± 6.2 mM (mean ± SD). In cells incubated in Na⁺-buffer [Na⁺]_i decreased to 3 ± 3.6 mM. If Na⁺/K⁺ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na⁺]_i increased to 47 ± 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetete, or thapsigargin had no effect on [Na⁺]_i. Incubating the cells in Ca²⁺-buffer rapidly increased [Na⁺]_i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca²⁺, nimodipine, or Ni²⁺ to these cells immediately decreased [Na⁺]_i, whereas Bay K 8644 enhanced the influx of Na⁺. In cells where [Na⁺]_i was increased the TRH-induced increase in intracellular free calcium ([Ca²⁺]_i) was decreased compared with control cells. Our results suggest that Na⁺ enters the cells via Ca²⁺ channels, and [Na⁺]_i may attenuate TRH-induced changes in [Ca²⁺]_i in GH₄C₁ cells. © 1993 Wiley-Liss, Inc.     

Impaired mitochondrial bioenergetics determines glutamate-induced delayed calcium deregulation in neurons

Background

Accumulation of glutamate in ischaemic CNS is thought to amplify neuronal death during a stroke. Exposure of neurons to toxic glutamate concentrations causes an initial transient increase in [Ca²⁺]_c followed by a delayed increase commonly termed delayed [Ca²⁺]_c deregulation (DCD).

Methods

We have used fluorescence imaging techniques to explore differences in glutamate-induced DCD in rat hippocampal neurons after different periods of time in culture (days in vitro; DIV).

Results

The amplitude of both the initial [Ca²⁺]_c signal and the number of cells showing DCD in response to glutamate increased with the duration of culture. The capacity of mitochondria to accumulate calcium in permeabilised neurons decreased with time in culture, although mitochondrial membrane potential at rest did not change. The rate of ATP consumption, measured as an increase in [Mg²⁺]_c following inhibition of ATP synthesis, was lower in ‘young’ neurons. The sensitivity of ‘young’ neurons to glutamate-induced DCD approximated to that of ‘old’ neurons when mitochondrial function was impaired using either FCCP or oligomycin. Further, following such treatment, cells showed a DCD-like response to increased [Ca²⁺]_c induced by KCl induced depolarisation which was never otherwise seen.

General significance

Thus, changes in cellular bioenergetics dictate the onset of DCD in response to glutamate.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

The role of cytoplasmic pH in the inhibitory action of high osmolarity on secretion from bovine adrenal chromaffin cells

Elevated osmolarity is known to inhibit secretion from a wide range of cells including bovine adrenal chromaffin cells. The mechanism of this inhibition is unclear but the elevated osmolarity has been proposed to oppose an osmotic driving force involved in exocytotic fusion. Using the fluorescent indicators quenel and fura2, we monitored the effect of elevated osmolarity on cytoplasmic pH (pH_i) and cytoplasmic free Ca²⁺ ([Ca²⁺]_i). Elevated osmolarity increased both pH_i and [Ca²⁺]_i, but had no effect on the [Ca²⁺]_i rise elicited by either K⁺ or nicotine. Elevating pH_i with NH₄Cl was shown to inhibit secretion from chromaffin cells. The elevation of pH_i by hyperosmolar solutions is proposed as one of the mechanisms by which elevated osmolarity inhibits secretion.     

Mechanisms of calcium transport in the giant axon of the squid and their physiological role

The giant axon of the squid has been extensively used as a model for studying Ca regulation in excitable cells. Different techniques (extrusion, injection and dialysis) have been employed to characterize Ca fluxes across the axon membrane. Since both Ca efflux and influx are markedly dependent on [Ca²⁺]_i, considerable effort has been dedicated to determine the resting value of the [Ca²⁺]_i. Results from different laboratories indicate that the [Ca²⁺]_i, in a normal fibre, range from 20–100 nM. Under dialysis conditions (internal control), with an imposed [Ca²⁺]_i of 80 nM, Ca influx is balanced by an outward Ca movement of about 40 f/CS. Ca extrusion occurs through two parallel transport systems: one having a high affinity for [Ca²⁺]_i, dependent on ATP, not affected by Na_i, Na_o, Ca_o, Mg_o and inhibited by internal vanadate (uncoupled component), the other, more prominent at relatively high [Ca²⁺]_i, does not require ATP, is inhibited by Na_i activated by Na_o and not inhibited by vanadate. (Na_o-dependent component). The existence of these two systems provide the axon with an effective way to maintain in the long term a constant low [Ca²⁺]_i in spite of short term fluctuations due to increased Ca influx during nervous activity.     

Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor-Associated Pores in Dibutyryl Cyclic AMP-Differentiated NG108-15 Cells

Abstract: The effect of replacement of extracellular Na⁺ with N-methyl-d -glucamine (NMG) on P₂ receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) with an EC₅₀ value of 230 µM. Replacement of Na⁺ with NMG as well as removal of Mg²⁺ from the bathing buffer potentiated ethidium bromide uptake, [Ca²⁺]_i increase, and ⁴⁵Ca²⁺ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg²⁺ to limit the amount of ATP^4?, replacement of Na⁺ with NMG had no effect on the ATP-induced [Ca²⁺]_i increase but caused a markedly larger [Ca²⁺]_i increase when the calculated concentration of ATP^4? was >10 µM. The calculated EC₅₀ value for ATP^4? stimulation of the [Ca²⁺]_i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca²⁺ release was the major pathway for the ATP-induced [Ca²⁺]_i increase; both removal of Mg²⁺ and replacement of Na⁺ with NMG did not affect the action of ATP. These data suggest that ATP^4?-promoted pores are antagonized by Na⁺ and Mg²⁺ in dibutyryl cyclic AMP-differentiated NG108-15 cells.     

Calcium supply effects on wheat cultivars differing in salt resistance with special reference to leaf cytosol ion homeostasis

Salinity causes changes in cytosolic Ca²⁺, [Ca²⁺]_cyt, Na⁺, [Na⁺]_cyt and pH, pH_cyt, which induce specific reactions and signals. Reactions causing a rebalancing of the physiological homeostasis of the cytosol could result in plant resistance and growth. Two wheat cultivars, Triticum aestivum, Seds1 and Vinjett, were grown in nutrient solution for 7 days under moderate salinity (0 and 50 mM NaCl) with and without extra addition of 5 mM CaSO₄ to investigate the seedling‐ion homeostasis under salinity. In the leaf protoplasts [Ca²⁺]_cyt, [Na⁺]_cyt and pH_cyt were detected using acetoxymethyl esters of the ion‐specific dyes, Fura 2, SBFI and BCECF, respectively, and fluorescence microscopy. In addition, both cultivars were grown for 3 weeks at 0, 50 and 125 mM NaCl with, or without, extra addition of 5 mM CaSO₄ to detect overall Na⁺ and Ca²⁺ concentrations in leaves and salinity effects on dry weights. In both cultivars, salinity decreased [Ca²⁺]_cyt, while at extra Ca²⁺ supplied, [Ca²⁺]_cyt increased. The [Ca²⁺]_cyt increase was accompanied by increase in the overall Ca²⁺ concentrations in leaves and decrease in the overall Na⁺ concentration. Moreover, irrespective of Ca²⁺ treatment under salinity, the cultivars reacted in different ways; [Na⁺]_cyt significantly increased only in cv. Vinjett, while pH_cyt increased only in cv. Seds1. Even at rather high total Na⁺ concentrations, the cytosolic concentrations were kept low in both cultivars. It is discussed whether the increase of [Ca²⁺]_cyt and pH_cyt can contribute to salt tolerance and if the cytosolic changes are due to changes in overall Ca²⁺ and Na⁺ concentrations.     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes

Activation of Na⁺-H⁺ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). We determined whether the change in [Ca²⁺]_i was secondary to the uptake of Na⁺, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na⁺] by treating the cells with ouabain or gramicidin failed to affect [Ca²⁺]_i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH₃, significantly elevated [Ca²⁺]_i. These results suggest an important role of cytoplasmic pH in the control of [Ca²⁺]_i in lymphocytes.     

A Model of Na/H Exchanger and Its Central Role in Regulation of pH and Na in Cardiac Myocytes

A new kinetic model of the Na⁺/H⁺ exchanger (NHE) was developed by fitting a variety of major experimental findings, such as ion-dependencies, forward/reverse mode, and the turnover rate. The role of NHE in ion homeostasis was examined by implementing the NHE model in a minimum cell model including intracellular pH buffer, Na⁺/K⁺ pump, background H⁺, and Na⁺ fluxes. This minimum cell model was validated by reconstructing recovery of pH_i from acidification, accompanying transient increase in [Na⁺]_i due to NHE activity. Based on this cell model, steady-state relationships among pH_i, [Na⁺]_I, and [Ca²⁺]_i were quantitatively determined, and thereby the critical level of acidosis for cell survival was predicted. The acidification reported during partial blockade of the Na⁺/K⁺ pump was not attributed to a dissipation of the Na⁺ gradient across the membrane, but to an increase in indirect H⁺ production. This NHE model, though not adapted to the dimeric behavioral aspects of NHE, can provide a strong clue to quantitative prediction of degree of acidification and accompanying disturbance of ion homeostasis under various pathophysiological conditions.     

Imipramine inhibition of TRPM‐like plasmalemmal Mg2+ transport in vascular smooth muscle cells

Depression is associated with vascular disease, such as myocardial infarction and stroke. Pharmacological treatments may contribute to this association. On the other hand, Mg²⁺ deficiency is also known to be a risk factor for the same category of diseases. In the present study, we examined the effect of imipramine on Mg²⁺ homeostasis in vascular smooth muscle, especially via melastatin‐type transient receptor potential (TRPM)‐like Mg²⁺‐permeable channels. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using ³¹P‐nuclear magnetic resonance (NMR) in porcine carotid arteries that express both TRPM6 and TRPM7, the latter being predominant. pH_i and intracellular phosphorus compounds were simultaneously monitored. To rule out Na⁺‐dependent Mg²⁺ transport, and to facilitate the activity of Mg²⁺‐permeable channels, experiments were carried out in the absence of Na⁺ and Ca²⁺. Changing the extracellular Mg²⁺ concentration to 0 and 6 mM significantly decreased and increased [Mg²⁺]_i, respectively, in a time‐dependent manner. Imipramine statistically significantly attenuated both of the bi‐directional [Mg²⁺]_i changes under the Na⁺‐ and Ca²⁺‐free conditions. This inhibitory effect was comparable in influx, and much more potent in efflux to that of 2‐aminoethoxydiphenyl borate, a well‐known blocker of TRPM7, a channel that plays a major role in cellular Mg²⁺ homeostasis. Neither [ATP]_i nor pH_i correlated with changes in [Mg²⁺]_i. The results indicate that imipramine suppresses Mg²⁺‐permeable channels presumably through a direct effect on the channel domain. This inhibitory effect appears to contribute, at least partially, to the link between antidepressants and the risk of vascular diseases.