Cytosolic Buffering Capacity Research Articles

Cytosolic Ca Buffering of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Adult Rabbit Ventricular Cardiomyocytes

A major concern about using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) for human disease modeling is their immature phenotype which may result in substantial differences in their physiology when compared to native cardiomyocytes. Here we compare intracellular Ca buffering of hiPSC-CM derived from healthy humans to that of acutely-isolated adult rabbit ventricular CM. hiPSC-CM (21 days post cardiac induction) were plated at low density on matrigel-coated dishes and studied 3-5 days after plating. While hiPSC were significantly smaller than rabbit CM (cell capacity 19±2 pF vs 101±12 pF, p<0.01) and had disorganized contractile filaments on electron microscopy, hiPSC-CM nevertheless exhibited robust troponin C expression, the major cytosolic Ca binding site. To measure cytosolic Ca buffering (Trafford method), caffeine (10 mM) was rapidly applied to release Ca from the SR, which then extruded via the NaCa exchanger (NCX), generating an inward NCX current. Integration of NCX current yielded the total amount of Ca released from the SR. Then Δ[Ca]total was plotted as a function of Δ[Ca]free and maximal cytosolic buffering capacity (Bmax) and Kd calculated. Cytosolic Ca buffering parameters were not significantly different between hiPSC-CM (Bmax=100±14μmol/Lcytosol, Kd=0.61±0.09 μM, n=25) and rabbit-CM (Bmax=117±15 μmol/Lcytosol, Kd=0.61±0.11 μM, n=9). EMD57033 (3 μM), which binds to troponin C and increases cytosolic Ca binding in murine CM, also increased cytosolic Ca binding affinity in hiPSC-CM (Kd=0.39±0.05 μM; p<0.05) without affecting Bmax (96±13 μmol/Lcytosol). Importantly, rates of non-NCX mediated Ca removal were not different between hiPSC-CM and adult rabbit CM. These results demonstrate that hiPSC-CM have cytosolic Ca buffering parameters similar to those of acutely-isolated native CM and therefore could be used for modeling cardiac diseases with altered myofilament Ca binding. Supported by AHA-12POST12080080(DK), NIH-RO1-HL71670(BCK).

PH regulation in an acidophilic green alga – a quantitative analysis

Short-term cytosolic pH regulation has three components: H(+) binding by buffering groups; H(+) transport out of the cytosol; and H(+) transport into the vacuole. To understand the large differences plants show in their tolerance to acidic environments, these three components were quantified in the acidophilic unicellular green alga Eremosphaera viridis. Intracellular pH was recorded using ion-selective microelectrodes, whereas constant doses of weak acid were applied over different time intervals. A mathematical model was developed that describes the recorded cytosolic pH changes. Recordings of cytosolic K(+) and Na(+) activities, and application of anion channel inhibitors, revealed which ion fluxes electrically compensate H(+) transport. The cytosolic buffer capacity was beta = 30 mM. Acidification resulted in a substantial and constant H(+) efflux that was probably driven by the plasmalemma H(+)-ATPase, and a proportional pH regulation caused by H(+) pumped into the vacuole. Under severe cytosolic acidification (> or = 1 pH) more than 50% of the ATP synthesized was used for H(+) pumping. While H(+) influx into the vacuole was compensated by cation release, H(+) efflux out of the cell was compensated by anion efflux. The data presented here give a complete and quantitative picture of the ion fluxes during acid loading in an acidophilic green plant cell.

Cytosolic pH buffering during exercise and recovery in skeletal muscle of patients with McArdle’s disease

Cellular pH control is important in muscle physiology, and for interpretation of (31)P magnetic resonance spectroscopy (MRS) data. Cellular acidification in exercise results from coupled glycolytic ATP production mitigated by cytosolic buffering, 'consumption' of H(+) by phosphocreatine (PCr) breakdown, and membrane transport processes. Ex vivo methods for cytosolic buffer capacity are vulnerable to artefact, and MRS methods often require assumptions. (31)P MRS of early exercise, when pH increases unopposed by glycolysis, is conceptually simple, but limited in normal muscle by time resolution and signal-to-noise. A therapeutic trial (Martinuzzi A et al. Musc Nerve 37: 350-357, 2007) in McArdle's disease (glycogen phosphorylase deficiency), where pH does not decrease with exercise, offered the opportunity to test (31)P MRS data obtained throughout incremental plantar flexion exercise and recovery in ten McArdle's patients against the simple model of cellular pH control. Changes in pH, [Pi] and [PCr] throughout exercise and recovery were quantitatively consistent with mean +/- SEM buffer capacity of 10 +/- 1 mM/(pH unit), which was not significantly different from the control subjects under the initial-exercise conditions where the comparison could be made. The simple model of cellular acid-base balance therefore gives an adequate account of cellular pH changes during both exercise and recovery in McArdle's disease.

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin(28kDa). Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin(28kDa)-null mutants. An unchanged rate of calcium transport was verified by measurements of (45)Ca incorporation into developing teeth in vivo. In enamel-forming cells, the absence of calbindin(28kDa) was not compensated by other cytosolic calcium-binding proteins as detectable by (45)Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.

Facilitated Lactate Transport by MCT1 when Coexpressed with the Sodium Bicarbonate Cotransporter (NBC) in Xenopus Oocytes

Monocarboxylate transporters (MCT) and sodium-bicarbonate cotransporters (NBC) transport acid/base equivalents and coexist in many epithelial and glial cells. In nervous systems, the electroneutral MCT1 isoform cotransports lactate and other monocarboxylates with H +, and is believed to be involved in the shuttling of energy-rich substrates between astrocytes and neurons. The NBC cotransports bicarbonate with sodium and generates a membrane current. We have expressed these transporter proteins, cloned from rat brain (MCT1) and human kidney (NBC), alone and together, by injecting the cRNA into oocytes of the frog Xenopus laevis, and measured intracellular pH changes and membrane currents under voltage-clamp with intracellular microelectrodes, and radiolabeled lactate uptake into the oocytes. We determined the cytosolic buffer capacity, the H + and lactate fluxes as induced by 3 and 10 mM lactate in oocytes expressing MCT1 and/or NBC, and in water-injected oocytes, in salines buffered with 5 mM HEPES alone or with 5% CO 2/10 mM HCO 3 − (pH 7.0). In MCT1 + NBC- but not in MCT1- or NBC-expressing oocytes, lactate activated a Na +- and HCO 3 −-dependent membrane current, indicating that lactate/H + cotransport via MCT1, due to the induced pH change, stimulates NBC activity. Lactate/H + cotransport by MCT1 was increased about twofold when MCT1 was expressed together with NBC. Our results suggest that the facilitation of MCT1 transport activity is mainly due to the increase in apparent buffer capacity contributed by the NBC, and thereby suppresses the build-up of intracellular H + during the influx of lactate/H +, which would reduce MCT1 activity. Hence these membrane transporters functionally cooperate and are able to increase ion/metabolite transport activity.

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes.

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.

Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.

Free [Ca2+] in agonist-sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA-AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre-loading the cells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA-AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+].

Mechanism of pyrithione-induced membrane depolarization in Neurospora crassa.

Pyrithione is a general inhibitor of membrane transport in fungi and is widely used in antidandruff shampoos as an antifungal agent. An electrophysiological approach has been used to determine the mode of action of pyrithione on the plasma membrane of the model ascomycete, Neurospora crassa. At pH 5.8, pyrithione induces a dramatic dose-dependent electrical depolarization of the membrane which is complete within 4 min, amounts to 110 mV at saturating pyrithione concentrations, and is half maximal between 0.6 and 0.8 mM pyrithione. Zinc pyrithione induces a similar response but exerts a half-maximal effect at around 0.3 mM. The depolarization is strongly dependent on external pH, being almost absent at pH 8.2, at which the concentration of the uncharged form of pyrithione--which might be expected to permeate the membrane freely--is markedly lowered. However, quantitative considerations based on cytosolic buffer capacity, the pKa of pyrithione, and the submillimolar concentration at which it is active appear to preclude significant cytosolic acidification on dissociation of the thiol proton from the uncharged form of pyrithione. Current-voltage analysis demonstrates that the depolarization is accompanied by a decrease in membrane electrical conductance in a manner consistent with inhibition of the primary proton pump and inconsistent with a mode of action of pyrithione on plasma membrane ion channels. We conclude that pyrithione inhibits membrane transport via a direct or indirect effect on the primary proton pump which energizes transport and that the site of action of pyrithione is likely to be intra- rather than extracellular.

The Na +/H + exchanger in transitional cells of the inner ear

The cytosolic pH (pH i) of transitional cells from the ampulla of the gerbil was measured micro-fluorometrically with the pH-sensitive dye 2′,7′-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) to assess the possible contribution of a Na +/H + exchanger to the regulation of H i. All experiments were conducted in virtually HCO 3 −free solutions. Under control conditions, pH i was 7.19 and addition of 10 −5 M ethylisopropylamiloride (EIPA), a blocker of Na +/H + exchange, caused a small but significant acidification of pH i. A transient exposure to 21.4 mM NH 4Cl caused a rapid cytosolic alkalinization followed by a brisk acidification and prompt recovery of pH i to its control value. The cytosolic buffer capacity (B i) was determined in the absence of Na + from changes in pH i which were elicited by [NH + 4] steps. B i was 4.7 mM/pH at pH i 7.19 and varied with pH i. The initial net proton flux J H, representative of Na +/H + exchange activity, was calculated from the product of the initial rate of alkalinization after an NH + 4-prepulse and B i at the corresponding pH i. J H was dependent on the extracellular Na + with an apparent K m of 64 mM, sensitive to the cytosolic [H +] with an apparent K m of 2.7 ∗ 10 −7 M (i.e. pH 6.6), and inhibited by EIPA with an IC 50 of 5 ∗ 10 −7 M. These data suggest that transitional cells contain in the basolateral membrane a Na +/H + exchanger of the amiloride-sensitive subtype.

Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO 3 ? /Cl? exchange

We have estimated the changes in cytosolic pH (pHi) that occur when human platelets are stimulated by thrombin. Changes in pHi were estimated (i) from the H+ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H+ efflux that averaged 7.7 +/- 1.6 mumol/10(11) platelets (means +/- SD) leading to an increase in pHi, from 7.05 +/- 0.04 to 7.45 +/- 0.05. Both H+ efflux and pHi changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4'-acetamido 4'-isothiostilbene-2,2'-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO3-/Cl- exchange. Removal of HCO3- or Cl- from the suspending buffer had no effect on the extent of the rise in pHi. After blockade of Na+/H+ exchange by 100 microM ethylisopropylamiloride (EIPA), thrombin induced a decrease in pHi the rate of which averaged 0.39 unit/min in HCO3(-)-containing medium, and 0.57 unit/min in HCO3(-)-free medium. The cytosolic buffer capacity for H+ was determined by the nigericin/NH4Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO3-, but only 17.2 mmol/(1xpH) in HCO3(-)-free buffer. The total amount of H+ transferred by Na+/H+ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO3- and to 14 mmol/l platelet cytosol in the presence of HCO3-, and is in good agreement with the estimated amount of Na+ uptake by ADP-stimulated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)