Effects of calcium and lanthanum on phosphate efflux from nonmyelinated nerve fibers

Summary Phosphate efflux was measured as the fractional rate of loss of radioactivity from rabbit vagus loaded with radiophosphate. The effects of changes in extracellular calcium and of lanthanum have been investigated. In Locke solution with normal, 0.9mm, calcium and without phosphate, the fractional rate of loss was 1.62×10^–3 min^–1 at 120 min after the beginning of the washing period and fell slowly (9% hr^–1) during washing from 2 to 6 hr. Addition of calcium to the Locke solution produced a transient increase followed by a reversible maintained increase in phosphate efflux. The latter was 40 and 75% above efflux in normal calcium for 20 and 50mm calcium, respectively. Removal of calcium, with or without addition of EGTA, produced only a transient increase in phosphate efflux, with no subsequent maintained change. Addition of low concentrations of lanthanum produced a reversible inhibition of phosphate efflux. Half-maximal inhibition was at 3.5 m lanthanum and appeared to be due to binding of lanthanum to more than one, probably two, sites. Measurements of inhibition by lanthanum at different calcium concentrations did not indicate any competition between calcium and lanthanum. It is suggested that at least a part of phosphate efflux depends on internal calcium and that lanthanum acts by preventing release of phosphate from the phosphate transport mechanism.     

Calcium efflux and intracellular exchangeable calcium in mammalian nonmyelinated nerve fibers

Summary Calcium efflux was measured in desheathed rabbit vagus nerves loaded with⁴⁵Ca²⁺. The effects of extracellular calcium, sodium, phosphate, potassium and lanthanum ions on the calcium efflux were investigated and the distribution of intracellular calcium determined by kinetic analysis of⁴⁵Ca²⁺ efflux profiles. The⁴⁵Ca²⁺ desaturation curve can be adequately described by three exponential terms. The rate constant of the first component (0.2 min^–1) corresponds to an efflux from an extracellular compartment. The two slow components had rate constants of 0.03 and 0.08 min^–1 and represent the efflux from two intracellular pools. The amounts of exchangeable calcium in these two pools, after a loading period of 150 min, were 0.170 and 0.102 mmol/kg wet weight, respectively. The total calcium efflux in physiological conditions amounted to about 24 fmol cm^–2 sec^–1. The magnitude of the two intracellular compartments as well as the total calcium efflux were markedly affected by extracellular phosphate, sodium and lanthanum, whereas the corresponding rate constants remained almost unchanged. Phosphate reversed the effect of sodium withdrawal on the calcium efflux: in the absence of phosphate, sodium withdrawal increased the calcium efflux to 224%, but in the presence of phosphate, sodium withdrawal decreased calcium efflux to 44%. Phosphate also affected the increase in calcium efflux produced by inhibitors of mitochondrial calcium uptake, suggesting that two different mitochondrial pools contribute to the control and regulation of intracellular calcium and of the transmembrane calcium transport.Deceased 18 April 1988     

Role of potassium in the phosphate efflux from mammalian nerve fibers

Summary The efflux of phosphate was measured in rabbit vagus nerve loaded with radiophosphate. The efflux was found to depend on the K concentration of the bathing solutions; increasing the K from 5.6 up to 150mm produced a maximal lowering of 28%; K-free solution produced a transient increase whose peak was 86% above the normal efflux. In the presence of Na, the K-free effect could be repeated; in Na-free solution, it was found only for the first application of the K-free solution. The phosphate efflux was not altered when K was replaced by Rb; replacement with Cs showed that this ion only partially mimics the effect of K.The results suggest that the transient increase in phosphate efflux is due to release of label from a K-dependent saturable binding site, which is distinct from the main intracellular pool. The binding site appears to be labeled from the inside by the Na-dependent phosphate efflux previously described. It may correspond to the phosphorylation of membrane phospholipids. A mathematical model of this system is developed and curves simulated by an analog computer are compared to the experimental results.Measurements of the membrane potential and the internal inorganic phosphate showed that the effect of K on the phosphate efflux could not be explained by changes in the membrane potential or in the internal phosphate pool.     

Effect of sodium on cellular calcium transport in rat kidney

Summary The influence of extracellular Na (Na _o ) on cellular Ca transport and distribution was studied in rat kidney slices. Calcium efflux from prelabeled slices was depressed when Na _o was completely replaced by choline or tetraethylammonium (TEA) ions and it was markedly stimulated when Na was reintroduced in a Na-free medium. However, reducing Na _o (with choline or TEA as substituting ions) did not increase the total slice⁴⁰Ca, their total exchangeable Ca pool, or the⁴⁰Ca or⁴⁵Ca of mitochondria isolated from these slices. Kinetic analyses of steady-state⁴⁵Ca desaturation curves showed that reducing Na _o depressed the exchange of Ca across the plasma membrane, slightly decreased the cytosolic Ca pool, but did not significantly affect the mitochondrial Ca pool and Ca cycling. Ouabain (10^–3 m) which should reduce the Na gradient across the plasma membrane had no effect on calcium distribution and transport. These results suggest that in kidney cells low Na _o depresses Ca influx as well as Ca efflux; there may be an interaction between Na and Ca at a possible carrier located in the plasma membrane, but there is no Na/Ca exchange as described in several excitable tissues.     

Role of intracellular sodium activity in the control of contraction in cardiac purkinje fibers

The role of intracellular sodium activity (a _Na ⁱ ) in the control of force was studied in sheep cardiac Purkinje fibers exposed to norepinephrine (NE) and high [Ca]_o in the absence and presence of overdrive or of a low concentration of strophanthidin. Both NE and high [Ca]_o decrease a _Na ⁱ and increase force, while overdrive increases and low strophanthidin decreases both parameters. In the presence of NE, overdrive increases a _Na ⁱ less than force and is followed by a more pronounced undershoot in a _Na ⁱ and force. In contrast, in high [Ca]_o overdrive increases a _Na ⁱ more than force and is followed by a less pronounced undershoot in a _Na ⁱ and force than in NE. High [Ca]_o increases force to a peak, but then the decreasing a _Na ⁱ reduces force. In all these conditions, a _Na ⁱ determines force changes during recovery from overdrive. NE and high [Ca]_o decrease a _Na ⁱ less and increase force more in low strophanthidin. Thus, changes in a _Na ⁱ modulate the increase in force due to increased Ca influx and control force development when Ca influx is either unchanged (low strophanthidin) or has reached a steady state (high [Ca]_o, recovery from overdrive).     

Continuous measurement of calcium influx in mammalian nonmyelinated nerve fibers: Effects of Na o,Ca o,and electrical activity

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in hyperosmotic depolarizing solutions. At the internal potentials obtained, K⁺ passes mainly through the inward rectifier potassium channels.Inhibition of K⁺ efflux by external Zn²⁺ (0.25 to 15mm) differs in three significant ways from inhibition by Ba²⁺. (1) The dose-response relation does not correspond to action at a single site. (2) The Zn²⁺-sensitivity of K⁺ efflux does not depend on [K⁺] _o at constant internal potential. (3) Zn²⁺ inhibition is reduced by hydrogen ions, while Ba²⁺ inhibition is unaffected. Further, the Ba²⁺-sensitivity of K⁺ efflux is not altered by a half-inhibiting Zn²⁺ concentration, suggesting that the two ions do not interact at a common site.The histidine-modifying reagent diethylpyrocarbonate (DEPC) reduces Zn²⁺ inhibition. After DEPC treatment Zn²⁺ inhibition is further reduced by low pH. DEPC has little effect on Ba²⁺ inhibition. Zn²⁺ inhibition is not altered by treatment with the sulfhydryl reagents 5,5-dithio-bis(2-nitrobenzoic acid) or dithiothreitol.The results can be described by either of two models in which two sites can bind Zn²⁺ and one or both of the sites may also bind H⁺. When both sites bind Zn²⁺, K⁺ efflux is inhibited, and a third site may then bind H⁺. The effects of DEPC can be accounted for by a decrease in H⁺ affinity of the first two sites by a factor of 50, and a decrease in Zn²⁺ affinity of these sites and of the H⁺ affinity of the third site by about one order of magnitude.     

Release of intracellular calcium stores leads to retraction of membrane sheets and cell death in mature mouse oligodendrocytes

The ability of mature oligodendrocytes (OLs) to recover from insult is important in repair of damage following demyelination. Since regulation of Ca²⁺ levels within cells plays a critical role in function and survival, this study investigates the effects of changes in cytoplasmic Ca²⁺ on the viability of cultured mouse OLs and their ability to maintain membrane sheets. Mature OLs in culture respond rapidly to the calcium ionophore A23187 and promptly return to resting Ca²⁺ levels when the ionophore is removed. Longer exposure to 0.1–1.0 μM A23187 leads to microtubule disruption, membrane sheet retraction and eventual cell death; nuclear lysis occurs in many of the OLs, as reported by Scolding, et al. (1) for rat OLs. In our cultures, mature OLs were more susceptible to nuclear lysis than were immature OLs or astroglia. Release of intracellular Ca²⁺ stores with thapsigargin at 5–10 μM also leads to retraction of membrane sheets. Following 6 hours of continuous exposure to thapsigargin, the effects on membrane sheets are reversed over the next 12 hours. After 18 hours of continuous exposure to thapsigargin, only occasional nuclear lysis is observed, but a number of the mature OLs show signs of DNA fragmentation, indicating that apoptotic death is occurring. Our results suggest that mature OLs cannot survive a prolonged influx of extracellular calcium as readily as immature OLs and astroglia, but have mechanisms to withstand similar increases in cytoplasmic Ca²⁺ following sustained release of intracellular stores. Special issue dedicated to Dr. Marion E. Smith.     

Mode of action of theophylline on sodium efflux in barnacle muscle fibers

Summary The response of the Na efflux in unpoisoned barnacle fibers to 10mm theophylline is biphasic; i.e., inhibition is followed by stimulation. The stimulatory response is unaffected by ouabain. Fibers pretreated with ouabain show no transitory inhibition when 10mm theophylline is applied, but show prompt stimulation the magnitude of which is comparable to that observed with unpoisoned fibers. The same holds true for lower concentrations of theophylline. Prior injection of 500mm EGTA completely abolishes the biphasic action of 10mm theophylline. External application of 10mm theophylline following removal of external Ca²⁺ fails to bring about a biphasic effect. Ca²⁺ restoration, however, results in a moderate rise in the Na efflux. External application of 10mm theophylline stimulates the Na efflux into Ca²⁺-free artificial seawater (ASW) when the test fibers are pretreated with ouabain. Injection of the protein inhibitor of Walsh leads to reduced stimulation by 10mm theophylline of the Na efflux in unpoisoned fibers. Injection of the protein inhibitor of Corbin into unpoisoned fibers leads to reduced stimulation by 10mm theophylline. Injection of cAMP into ouabainpoisoned fibers, following internal application of Corbin's inhibitor and external application of 10mm theophylline, fails to cause a marked rise in the ouabain-insensitive Na efflux. Injection of Corbin's inhibitor into ouabain-poisoned fibers, following the onset of peak stimulation by 10mm theophylline, fails to reduce the Na efflux. Fibers injected with 1mm and 100mm EGTA and exposed to 10mm theophylline show a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP when the concentration of theophylline is 10mm. A poor response to injected cAMP is also seen in fibers bathed in Ca-free ASW containing 10mm theophylline. Theophylline (10mm) fails to cause an enhanced stimulation of the ouabain-insensitive Na efflux into Ca-free 3mm-HEPES ASW or 10mm-Ca²⁺-3mm-HEPES ASW following the addition of protons to the bathing medium. An enhanced response is similarly not observed with injected cAMP following the addition of theophylline to the bathing medium. Injection of 8-fluorotheophylline, 3-isobutyl-1-methylxanthine and doxantrazole leads to a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP. Contraction always takes place upon injecting these substances. These results are in keeping with the theory that theophylline acts chiefly by reducing myoplasmic pCa (pCa-log₁₀[Ca²⁺]), and that a reduced pCa leads to stimulation of the ouabain-insensitive Na efflux as the result of activation of the cGMP-dependent protein kinase system by newly formed cGMP.     

Stimulation by high external potassium of the sodium efflux in barnacle muscle fibers

Summary Single barnacle muscle fibers fromBalanus nubilus were used to study the effect of elevated external potassium concentration, [K] _o , on Na efflux, membrane potential, and cyclic nucleotide levels. Elevation of [K] _o causes a prompt, transient stimulation of the ouabain-insensitive Na efflux. The minimal effective concentrations is 20mm. The membrane potential of ouabain-treated fibers bathed in 10mm Ca²⁺ artificial seawater (ASW) or in Ca²⁺-free ASW decreases approximately linearly with increasing logarithm of [K] _o . The slope of the plot is slightly steeper for fibers bathed in Ca²⁺-free ASW. The magnitude of the stimulatory response of the ouabain-insensitive Na efflux to 100mmK _o depends on the external Na⁺ and Ca²⁺ concentrations, as well as on external pH, but is independent of external Mg²⁺ concentration. External application of 10^–4 m verapamil virtually abolishes the response of the Na efflux to subsequent K-depolarization. Stabilization of myoplasmic-free Ca²⁺ by injection of 250mm EGTA before exposure of the fiber to 100mm K _o leads to 60% reduction in the magnitude of the stimulation. Pre-injection of a pure inhibitor of cyclic AMP-dependent protein kinase reduces the response of the Na efflux to 100mm K _o by 50%. Increasing intracellular ATP, by injection of 0.5m ATP-Na₂ before elevation of [K] _o , fails to prolong the duration of the stimulation of the Na efflux. Exposure of ouabain-treated, cannulated fibers to 100mm K _o for time periods ranging from 30 sec to 10 min causes a small (60%), but significant, increase in the intracellular content of cyclic AMP with little change in the cyclic GMP level. These results are compatible with the view that the stimulatory response of the ouabain-insensitive Na efflux to high K _o is largely due to a fall in myoplasmicpCa resulting from activation of voltage-dependent Ca²⁺ channels and that an accompanying rise in internal cAMP accounts for a portion of this response.     

Sodium inward currents through calcium channels in mealworm muscle fibers

The contribution of Na⁺ ions to the nonsynaptic electrogenesis was studied in the larval muscle fibers of mealworm, Tenebrio molitor, using currentclamp and voltage-clamp techniques. Na-dependent graded responses were generated by depolarizing current stimuli in Ca²⁺-free solutions. These responses were insensitive to tetrodotoxin and were blocked by Co²⁺. Large inward-going currents were elicited by step depolarizations in Ca²⁺-free solutions under voltage-clamp conditions. The inward currents were totally eliminated by removal of Na⁺ from the bathing solution. These results indicate that the calcium channel of mealworm muscle is permeable to Na⁺.     

The role of an intracellular cysteine stretch in the sorting of the type II Na/phosphate cotransporter

The type II Na/phosphate cotransporters (NaPi-II) are critical for the control of plasma phosphate levels in vertebrates. NaPi-IIb mediates phosphate uptake from the small intestine followed by glomerular filtration and selective reabsorption from the renal proximal tubule by NaPi-IIa and NaPi-IIc. A C-terminal stretch of cysteine residues represents the hallmark of the NaPi-IIb isoforms. This motif is well conserved among NaPi-IIb type transporters but not found in other membrane proteins. To investigate the role of this motif we analyzed NaPi-II constructs in transiently and stably transfected MDCK cells. This cell line targets the NaPi-IIb isoforms from flounder and mouse to the apical membrane whereas the mouse IIa isoform shows no plasma membrane preference. Different parts of mouse NaPi-IIa and NaPi-IIb C-termini were fused to GFP-tagged flounder NaPi-II. The constructs showed strong staining of the plasma membrane with NaPi-IIb related constructs sorted predominantly apically, the IIa constructs localized apically and basolaterally with slight intracellular retention. When the cysteine stretch was inserted into the NaPi-IIa C-terminus, the construct was retained in a cytoplasmic compartment. 2-bromopalmitate, a specific palmitoylation inhibitor, released the transporter to apical and basolateral membranes. The drug also leads to a redistribution of the NaPi-IIb construct to both plasma membrane compartments. Immunoprecipitation of tagged NaPi-II constructs from [³H]-palmitate labeled MDCK cells indicated that the cysteine stretch is palmitoylated. Our results suggest that the modified cysteine motif prevents the constructs from basolateral sorting. Additional sorting determinants located downstream of the cysteine stretch may release the cargo to the apical compartment.     

Dent disease: A window into calcium and phosphate transport

This review examines calcium and phosphate transport in the kidney through the lens of the rare X‐linked genetic disorder Dent disease. Dent disease type 1 (DD1) is caused by mutations in the CLCN5 gene encoding ClC‐5, a Cl^?/H⁺ antiporter localized to early endosomes of the proximal tubule (PT). Phenotypic features commonly include low molecular weight proteinuria (LMWP), hypercalciuria, focal global sclerosis and chronic kidney disease; calcium nephrolithiasis, nephrocalcinosis and hypophosphatemic rickets are less commonly observed. Although it is not surprising that abnormal endosomal function and recycling in the PT could result in LMWP, it is less clear how ClC‐5 dysfunction disturbs calcium and phosphate metabolism. It is known that the majority of calcium and phosphate transport occurs in PT cells, and PT endocytosis is essential for calcium and phosphorus reabsorption in this nephron segment. Evidence from ClC‐5 KO models suggests that ClC‐5 mediates parathormone endocytosis from tubular fluid. In addition, ClC‐5 dysfunction alters expression of the sodium/proton exchanger NHE3 on the PT apical surface thus altering transcellular sodium movement and hence paracellular calcium reabsorption. A potential role for NHE3 dysfunction in the DD1 phenotype has never been investigated, either in DD models or in patients with DD1, even though patients with DD1 exhibit renal sodium and potassium wasting, especially when exposed to even a low dose of thiazide diuretic. Thus, insights from the rare disease DD1 may inform possible underlying mechanisms for the phenotype of hypercalciuria and idiopathic calcium stones.     

Identification and functional reconstitution of phosphate: Sugar phosphate antiport ofStaphylococcus aureus

Summary Resting cells ofStaphylococcus aureus displayed a phosphate (P_i) exchange that was induced by growth with glucose 6-phosphate (G6P) orsn-glycerol 3-phosphate (G3P). P_i-loaded membrane vesicles from these cells accumulated³²P_i, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of³²P_i (andl-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (dl-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both P_i anddl-lactate to establish an internal pool of P_i. Thistrans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal P_i.Reconstitution of membrane protein allowed a quantitative analysis of P_i-linked exchange. P_i-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous³²P_iP_i exchange (K _i's of 2.2 and 1.4mm;V _max's of 180 and 83 nmol P_i/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (K _i=27 m) over G3P (K _i=1.3mm) and P_i (K _i=2.2mm), suggesting that the same antiporter was induced in both cases. We conclude that³²P_iP_i exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate. In this respect, P_i-linked antiport inS. aureus resembles other examples in a newly described family of bacterial transporters that use anion exchange as the molecular basis of solute transport.     

Caffeine inhibition of calcium accumulation by the sarcoplasmic reticulum in mammalian skinned fibers

Summary Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca²⁺ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca²⁺ exceeds 0.3 to 0.4 m, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg²⁺ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca²⁺ concentration dependence of Ca uptake (K _0.5, 0.5 to 0.8 m; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum

Summary The localization of serotonin (5-HT)-immunoreactive nerve fibers in the cerebellum of the rat and cat was investigated by means of the peroxidase-anti-peroxidase (PAP) method using highly specific antibodies to 5-HT.Serotonin-containing nerve fibers were distributed throughout the entire cerebellum including the deep cerebellar nuclei, while 5-HT-positive neuronal somata were not detected in the cerebellum of either species. A different pattern of 5-HT innervation was found among the three layers of the cerebellar cortex. There were also interspecific differences in the pattern of distribution of 5-HT. In the rat, the pool of 5-HT nerve fibers mainly consisted of tangential elements, which were predominant in the molecular layer, while in the cat only a few 5-HT fibers were found in the molecular layer of the cerebellar cortex; dense networks of 5-HT nerve fibers were present in the granular layer. Some differences are evident in the pattern of distribution of 5-HT fibers in cerebellar regions classified on an anatomical and functional basis.This work was supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

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.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.