Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion

Background

Orthophosphate (P_i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P_i acquisition by Trypanosoma cruzi.

Methods

³²P_i influx was measured in T. cruzi epimastigotes. The expression of P_i transporter genes and the coupling of the uptake to Na⁺, H⁺ and K⁺ fluxes were also investigated. The transport capacities of different evolutive forms were compared.

Results

Epimastigotes grew significantly more slowly in 2 mM than in 50 mM P_i. Influx of P_i into parasites grown under low P_i conditions took place in the absence and presence of Na⁺. We found that the parasites express TcPho84, a H⁺:P_i-symporter, and TcPho89, a Na⁺:P_i-symporter. Both P_i influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na⁺-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of P_i. Valinomycin (K⁺ ionophore) or SCH28028 (inhibitor of (H⁺ + K⁺)ATPase) significantly inhibited P_i uptake, indicating that an inwardly-directed H⁺ gradient energizes uphill P_i entry and that K⁺ recycling plays a key role in P_i influx. Furosemide, an inhibitor of the ouabain-insensitive Na⁺-ATPase, decreased only the Na⁺-dependent P_i uptake, indicating that this Na⁺ pump generates the Na⁺ gradient utilized by the symporter. Trypomastigote forms take up P_i inefficiently.

Conclusions

P_i starvation stimulates membrane potential-sensitive P_i uptake through different pathways coupled to Na⁺ or H⁺/K⁺ fluxes.

General significance

This study unravels the mechanisms of P_i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.     

Na---Ca exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca²⁺ as essential activator of the Na⁺---Ca²⁺ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 μM free calcium exhibited a 2-fold increase in the initial rate of Na_i⁺-dependent Ca²⁺ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca²⁺ on the reverse mode of Na⁺---Ca²⁺ exchange (i.e. Na_i⁺---Ca₀²⁺ exchange) is saturated at about 100 μM Ca_i²⁺ and displays an apparent K_1/2 of 12 μM. Intravesicular Ca²⁺ produced activation of Na_i⁺---Ca₀²⁺ exchange activity rather than an increase in Ca²⁺ uptake due to Ca²⁺---Ca²⁺ exchange. The presence of Ca_i²⁺ was essential for the Na_i⁺-dependent Na⁺ influx, a partial reaction of the Na⁺---Ca²⁺ exchanger. In fact, the Na⁺ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca_i²⁺ an additional Na⁺---Na⁺ exchange was measured. The results suggest that in nerve membrane vesicles Ca²⁺ at the inner aspect of the membrane acts as an activator of the Na⁺---Ca²⁺ exchange system.     

Platelet Ca2+ handling in essential hypertension: Role of a plasma ouabain-like factor

A humoral ouabain-like plasma factor has been observed in patients with essential hypertension (EHT). In the present study, we hypothesized that this humoral factor might be responsible for the elevated cytosolic free calcium concentrations [Ca²⁺]_i seen in these patients. Patients with mild to moderate EHT and their normotensive first degree blood relatives (NTBR) participated in the study. Platelet Na⁺, K⁺-ATPase activity was assayed in EHT patients and their NT first-degree relatives. To confirm the ouabain-like activity in plasma from EHT patients, control platelets were incubated with EHT and NTBR plasma and their Na⁺, K⁺-ATPase activity was measured. In addition, the effect of EHT plasma on platelet⁴⁵Ca-uptake was studied. Thein vitro effects of ouabain (10 ΜM) on (i)⁴⁵Ca-uptake and (ii) [Ca²⁺]_i response in control platelets were also observed. A decreased Na⁺K⁺-ATPase activity (P< 0.05) was observed in platelet membranes from EHT patients. Incubation of control platelets with EHT plasma decreased their Na⁺, K⁺-ATPase activity (P< 0.01) and increased their⁴⁵Ca-uptake (P< 0.05). C-18 Sep-Pak filtered hypertensive plasma extracts (containing the ouabain-like fraction) also decreased Na⁺, K⁺-ATPase activity (P< 001) in control platelet membranes.In vitro incubation of control platelets with ouabain increased⁴⁵Ca-uptake (P< 005) and [Ca²⁺]_i response (P< 0.05) in these platelets. Thus it appears that an ouabain-like factor in the EHT plasma may contribute to the elevated platelet [Ca²⁺]_i observed in EHT patients.     

The modulation of the calcium pump of human red cells by Na+ and K+

1. The sidedness of Ca²⁺-pump activation by Na⁺ and K⁺ was studied by atomic absorption spectrophotometry in human erythrocyte ghosts, which had been prepared in dextran solutions and resealed to alkali cations. 2. When ghosts were incubated in an all-choline medium, the increase in Na_i⁺ elicited an inhibitory-stimulatory effect on Ca²⁺ extrusion. By contrast, only a stimulatory action was induced when choline was replaced by Na₀⁺. 3. A dual effect on active Ca²⁺ efflux was also produced by increasing K_i⁺ or K₀⁺. The biphasic response to the latter, however, was absent from high-K⁺ ghosts. Furthermore, the stimulation obtained at high K₀⁺ was additive to that elicited by K_i⁺. 4. The results suggest that Na⁺ and K⁺ stimulate the Ca²⁺ pump of human red cells through two different mechanisms. The first one appears to be an electric coupling between Ca²⁺ efflux and the external activating cation. The other seems associated with the molecular reactions of the Ca²⁺-pump protein.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Measuring Ca binding to short chain fatty acids and gluconate with a Ca electrode: Role of the reference electrode

Many organic anions bind free Ca²⁺, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (K_app) describing the Ca²⁺ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca²⁺ electrodes coupled to either a 3 M KCl or a Na⁺ selective electrode were used to redetermine K_app. All solutions contained 130 mM Na⁺, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl⁻ that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of K_app if left uncorrected. Because the Na⁺ concentration in all solutions was constant, a Na⁺ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of K_app either after correction for these LJP changes or via direct reference to a Na⁺ electrode showed that SCFAs do not bind Ca²⁺ and that the K_app for the binding of Ca²⁺ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM.     

Glucose Decouples Intracellular Ca2+ Activity from Glucagon Secretion in Mouse Pancreatic Islet Alpha-Cells

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca²⁺]_i) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca²⁺]_i and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K_ATP channel activity but not on tetrodotoxin-sensitive Na⁺ channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca²⁺]_i and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K_ATP channel activity or reduction in α-cell [Ca²⁺]_i. Our results demonstrate that glucose uncouples the positive relationship between [Ca²⁺]_i and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.     

A mathematical model of Ca2+ dynamics in rat mesenteric smooth muscle cell: agonist and NO stimulation

A mathematical model of calcium dynamics in vascular smooth muscle cell (SMC) was developed based on data mostly from rat mesenteric arterioles. The model focuses on (a) the plasma membrane electrophysiology; (b) Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR); (c) cytosolic balance of Ca²⁺, Na⁺, K⁺, and Cl⁻ ions; and (d) IP₃ and cGMP formation in response to norepinephrine (NE) and nitric oxide (NO) stimulation. Stimulation with NE induced membrane depolarization and an intracellular Ca²⁺ ([Ca²⁺]_i) transient followed by a plateau. The plateau concentrations were mostly determined by the activation of voltage-operated Ca²⁺ channels. NE causes a greater increase in [Ca²⁺]_i than stimulation with KCl to equivalent depolarization. Model simulations suggest that the effect of [Na⁺]_i accumulation on the Na⁺/Ca²⁺ exchanger (NCX) can potentially account for this difference. Elevation of [Ca²⁺]_i within a concentration window (150-300 nM) by NE or KCl initiated [Ca²⁺]_i oscillations with a concentration-dependent period. The oscillations were generated by the nonlinear dynamics of Ca²⁺ release and refilling in the SR. NO repolarized the NE-stimulated SMC and restored low [Ca²⁺]_i mainly through its effect on Ca²⁺-activated K⁺ channels. Under certain conditions, Na⁺-K⁺-ATPase inhibition can result in the elevation of [Na⁺]_i and the reversal of NCX, increasing resting cytosolic and SR Ca²⁺ content, as well as reactivity to NE. Blockade of the NCX's reverse mode could eliminate these effects. We conclude that the integration of the selected cellular components yields a mathematical model that reproduces, satisfactorily, some of the established features of SMC physiology. Simulations suggest a potential role of intracellular Na⁺ in modulating Ca²⁺ dynamics and provide insights into the mechanisms of SMC constriction, relaxation, and the phenomenon of vasomotion. The model will provide the basis for the development of multi-cellular mathematical models that will investigate microcirculatory function in health and disease.     

Thyroid hormones increase Na+–Pi co-transport activity in intestinal brush border membrane: Role of membrane lipid composition and fluidity

In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na⁺–P_i transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid composition and fluidity. Augmentation of net P_i balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of P_i transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats while Na⁺–P_i transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na⁺–P_i transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important role in modulating Na⁺–P_i transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005)     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Characterization of Na+-Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons

Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na⁺-dependent inorganic phosphate (P_i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na⁺-dependent ³²P_i cotransport in cultured fetal rat cortical neurons, where >90% of saturable P_i uptake is Na⁺-dependent. Saturable, Na⁺-dependent ³²P_i uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na⁺-dependent P_i transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent K_m values for P_i and Na⁺ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca²⁺ markedly reduced (>60%) Na⁺-dependent P_i uptake, with a threshold for maximal P_i import of 1–2.5 mM CaCl₂. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na⁺ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na⁺. Furthermore, a substantial fraction of the ³²P_i cotransported with Na⁺ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na⁺-dependent P_i import rates. These data establish the presence of a Na⁺-dependent P_i cotransport system in neurons of the CNS, demonstrate the Ca²⁺-dependent nature of ³²P_i uptake, and suggest that the neuronal Na⁺-dependent P_i cotransporter may import P_i required for the production of high-energy compounds vital to neuronal metabolism.     

Inhibition of plasma membrane Ca-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca-occluded state

The bidentate complex of ATP with Cr³⁺, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca²⁺-ATPase and the Na⁺,K⁺-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca²⁺ and Na⁺, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca²⁺-ATPase. The complex inhibited with similar efficiency the Ca²⁺-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T_1/2 = 30 min at 37 °C) with a K_i = 28 ± 9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg²⁺ but unaltered when Ca²⁺ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca²⁺ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La³⁺ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca²⁺ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca²⁺ promoted by the plasma membrane Ca²⁺-ATPase goes through an enzymatic phospho-intermediate that maintains Ca²⁺ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

Systematic characterization of the ionic basis of rabbit cellular electrophysiology using two ventricular models

Several mathematical models of rabbit ventricular action potential (AP) have been proposed to investigate mechanisms of arrhythmias and excitation-contraction coupling. Our study aims at systematically characterizing how ionic current properties modulate the main cellular biomarkers of arrhythmic risk using two widely-used rabbit ventricular models, and comparing simulation results using the two models with experimental data available for rabbit. A sensitivity analysis of AP properties, Ca²⁺ and Na⁺ dynamics, and their rate dependence to variations (±15% and ±30%) in the main transmembrane current conductances and kinetics was performed using the Shannon et al. (2004) and the [Mahajan et?al., 2008a] and [Mahajan et?al., 2008b] AP rabbit models. The effects of severe transmembrane current blocks (up to 100%) on steady-state AP and calcium transients, and AP duration (APD) restitution curves were also simulated using both models. Our simulations show that, in both virtual rabbit cardiomyocytes, APD is significantly modified by most repolarization currents, AP triangulation is regulated mostly by the inward rectifier K⁺ current (I_K1) whereas APD rate adaptation as well as [Na⁺]_i rate dependence is influenced by the Na⁺/K⁺ pump current (I_NaK). In addition, steady-state [Ca²⁺]_i levels, APD restitution properties and [Ca²⁺]_i rate dependence are strongly dependent on I_NaK, the L-Type Ca²⁺ current (I_CaL) and the Na⁺/Ca²⁺ exchanger current (I_NaCa), although the relative role of these currents is markedly model dependent. Furthermore, our results show that simulations using both models agree with many experimentally-reported electrophysiological characteristics. However, our study shows that the Shannon et al. model mimics rabbit electrophysiology more accurately at normal pacing rates, whereas Mahajan et al. model behaves more appropriately at faster rates. Our results reinforce the usefulness of sensitivity analysis for further understanding of cellular electrophysiology and validation of cardiac AP models.     

Functional asymmetry of bidirectional Ca-movements in an archaeal sodium–calcium exchanger (NCX_Mj)

Dynamic features of Ca²⁺ interactions with transport and regulatory sites control the Ca²⁺-fluxes in mammalian Na⁺/Ca²⁺(NCX) exchangers bearing the Ca²⁺-binding regulatory domains on the cytosolic 5L6 loop. The crystal structure of Methanococcus jannaschii NCX (NCX_Mj) may serve as a template for studying ion-transport mechanisms since NCX_Mj does not contain the regulatory domains. The turnover rate of Na⁺/Ca²⁺ exchange (k_cat = 0.5 ± 0.2s⁻¹) in WT–NCX_Mj is 10³–10⁴ times slower than in mammalian NCX. In NCX_Mj, the intrinsic equilibrium (K_int) for bidirectional Ca²⁺ movements (defined as the ratio between the cytosolic and extracellular K_m of Ca²⁺/Ca²⁺ exchange) is asymmetric, K_int = 0.15 ± 0.5. Therefore, the Ca²⁺ movement from the cytosol to the extracellular side is ∼7-times faster than in the opposite direction, thereby representing a stabilization of outward-facing (extracellular) access. This intrinsic asymmetry accounts for observed differences in the cytosolic and extracellulr K_m values having a physiological relevance. Bidirectional Ca²⁺ movements are also asymmetric in mammalian NCX. Thus, the stabilization of the outward-facing access along the transport cycle is a common feature among NCX orthologs despite huge differences in the ion-transport kinetics. Elongation of the cytosolic 5L6 loop in NCX_Mj by 8 or 14 residues accelerates the ion transport rates (k_cat) ∼10 fold, while increasing the K_int values 100–250-fold (K_int = 15–35). Therefore, 5L6 controls both the intrinsic equilibrium and rates of bidirectional Ca²⁺ movements in NCX proteins. Some additional structural elements may shape the kinetic variances among phylogenetically distant NCX variants, although the intrinsic asymmetry (K_int) of bidirectional Ca²⁺ movements seems to be comparable among evolutionary diverged NCX variants.     

Erratum to: Splicing factors differentially expressed in psoriasis alter mRNA maturation of disease-associated EDA+ fibronectin

Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous β-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe²⁺ and H⁺ cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na⁺/H⁺ exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na⁺/Ca²⁺ exchanger (NCX1) and plasma membrane Ca²⁺-ATPase (PMCA_1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in β-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.     

Effect of internal and external K+ on Na+−Ca2+ exchange in dialyzed squid axons under voltage clamp conditions

The effect of external and internal K⁺ on Na_o⁺-dependent Ca²⁺ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K⁺ (up to 70 mM) has no effect on Na⁺?Ca²⁺ exchange. Removal of K_i⁺ causes a marked inhibition in the Na_o⁺-dependent Ca²⁺ efflux component. Internal K⁺ activates the Na⁺?Ca²⁺ exchange with low affinity $\text{(K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 90 mM)}$. Activation by K_i⁺ is similar in the presence or in the absence of Na_i⁺, thus ruling out a displacement of Na_i⁺ from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca²⁺ efflux on K_i⁺. The present results demonstrate that K_i⁺ is an important cofactor (partially required) for the proper functioning of the forward Na⁺?Ca²⁺ exchange.     

A Transmural Gradient in the Cardiac Na/K Pump Generates a Transmural Gradient in Na/Ca Exchange

We previously demonstrated a transmural gradient in Na/K pump current (I _P) and [Na⁺] _i , with the highest maximum I _P and lowest [Na⁺] _i in epicardium. The present study examines the relationship between the transmural gradient in I _P and Na/Ca exchange (NCX). Myocytes were isolated from canine left ventricle. Whole-cell patch clamp was used to measure current generated by NCX (I _NCX) and inward background calcium current (I _ibCa), defined as inward current through Ca²⁺ channels less outward current through Ca²⁺-ATPase. When resting myocytes from endocardium (Endo), midmyocardium (Mid) or epicardium (Epi) were studied in the same conditions, I _NCX was the same and I _ibCa was zero. Moreover, Western blots were consistent with NCX protein being uniform across the wall. However, the gradient in [Na⁺] _i , with I _ibCa = 0, should create a gradient in [Ca²⁺] _i . To test this hypothesis, we measured resting [Ca²⁺] _i using two methods, based on either transport or the Ca²⁺-sensitive dye Fura2. Both methods demonstrated a significant transmural gradient in resting [Ca²⁺] _i , with Endo > Mid > Epi. This gradient was eliminated by exposing Epi to sufficient ouabain to partially inhibit Na/K pumps, thus increasing [Na⁺] _i to values similar to those in Endo. These data support the existence of a transmural gradient for Ca²⁺ removal by NCX. This gradient is not due to differences in expression of NCX; rather, it is generated by a transmural gradient in [Na⁺] _i , which is due to a transmural gradient in plasma membrane expression of the Na/K pump.     

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na/Ca exchange

Fluctuating extracellular Ca²⁺ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca²⁺ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O₂ consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca²⁺, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na⁺ and the subsequent activation of mitochondrial Na⁺/Ca²⁺ and Na⁺/H⁺ exchange. The mitochondrial Na⁺/Ca²⁺ exchanger (_mNCX) activated by Na⁺ influx reduces Ca²⁺ and increases Na⁺ levels in the mitochondrial matrix. The excess of Na⁺ activates the mitochondrial Na⁺/H⁺ exchanger (NHE) increasing the outward pumping of protons, electron transport and O₂ consumption. Reduction in extracellular Na⁺ and inhibition of Na⁺ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the _mNCX, L-type voltage gated Ca²⁺ channels or the release of Ca²⁺ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca²⁺ exchange is critical for response development.     

Cardiomyopathy associated with noninsulin-dependent diabetes

Summary Cardiovascular disease represents the major cause of morbidity and mortality in noninsulin-dependent diabetic patients. While it was once thought that atherosclerotic vascular disease was responsible for all of these adverse effects, recent studies support the notion that one of the major adverse complications of diabetes is the development of a diabetic cardiomyopathy characterized by defects in both diastolic and systolic function. Contributing to the development of the cardiomyopathy is a shift in myosin isozyme content in favor of the least active V₃ form. Also defective in the noninsulin-dependent diabetic heart is regulation of calcium homeostasis. While transport of calcium by the sarcolemmal and sarcoplasmic reticular calcium pumps are minimally affected by noninsulin-dependent diabetes, significant impairment occurs in sarcolemmal Na⁺-Ca²⁺ exchanger activity. This defect limits the ability of the diabetic heart to extrude calcium, contributing to an elevation in [Ca²⁺]_i. Also promoting the accumulation of calcium by the diabetic cell is a decrease in Na⁺, K⁺ ATPase activity, which is known to increase [Ca²⁺]_i secondary to a rise in [Na⁺]_i. In addition, calcium influx via the calcium channel is stimulated. Although the molecular mechanisms underlying these defects are presently unknown, the possibility that they may be related to aberrations in glucose or lipid metabolism are considered. The evidence suggests that classical theories of glucose toxicity, such as excessive polyol production or glycosylation, appear to be insignificant factors in heart. Also insignificant are defects in lipid metabolism leading to accumulation of toxic lipid amphiphiles or triacylglycerol. Rather, the major defects involve membrane changes, such as phosphatidylethanolamine N-methylation and protein phosphorylation, which can be attributed to the state of insulin resistance.