Functional diversity of electrogenic Na+–HCO3− cotransport in ventricular myocytes from rat, rabbit and guinea pig
Taku YamamotoPawel SwietachAlessandra RossiniShih‐Hurng LohRichard D. Vaughan‐JonesKenneth W. Spitzer
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The Na(+)-HCO(3)(-) cotransporter (NBC) is an important sarcolemmal acid extruder in cardiac muscle. The characteristics of NBC expressed functionally in heart are controversial, with reports suggesting electroneutral (NBCn; 1HCO(3)(-) : 1Na(+); coupling coefficient N= 1) or electrogenic forms of the transporter (NBCe; equivalent to 2HCO(3)(-) : 1Na(+); N= 2). We have used voltage-clamp and epifluorescence techniques to compare NBC activity in isolated ventricular myocytes from rabbit, rat and guinea pig. Depolarization (by voltage clamp or hyperkalaemia) reversibly increased steady-state pH(i) while hyperpolarization decreased it, effects seen only in CO(2)/HCO(3)(-)-buffered solutions, and blocked by S0859 (cardiac NBC inhibitor). Species differences in amplitude of these pH(i) changes were rat > guinea pig approximately rabbit. Tonic depolarization (-140 mV to -0 mV) accelerated NBC-mediated pH(i) recovery from an intracellular acid load. At 0 mV, NBC-mediated outward current at resting pH(i) was +0.52 +/- 0.05 pA pF(-1) (rat, n= 5), +0.26 +/- 0.05 pA pF(-1) (guinea pig, n= 5) and +0.10 +/- 0.03 pA pF(-1) (rabbit, n= 9), with reversal potentials near -100 mV, consistent with N= 2. The above results indicate a functionally active voltage-sensitive NBCe in these species. Voltage-clamp hyperpolarization negative to the reversal potential for NBCe failed, however, to terminate or reverse NBC-mediated pH(i)-recovery from an acid load although it was slowed significantly, suggesting electroneutral NBC may also be operational. NBC-mediated pH(i) recovery was associated with a rise of [Na(+)](i) at a rate approximately 25% of that mediated via NHE, and consistent with an apparent NBC stoichiometry between N= 1 and N= 2. In conclusion, NBCe in the ventricular myocyte displays considerable functional variation among the three species tested (greatest in rat, least in rabbit) and may coexist with some NBCn activity.Keywords:
Hyperpolarization
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Intracellular pH
We have examined the hypothesis that a regulatory interplay between pH-regulated plasma membrane K+ conductance (gK+) and electrogenic Na+/HCO3- cotransport contributes importantly to regulation of intracellular pH (pHi) in hepatocytes. In individual cells, membrane depolarization produced by transient exposure to 50 mM K+ caused a reversible increase in pHi in the presence, but not absence, of HCO3-, consistent with voltage-dependent HCO3- influx. In the absence of HCO3-, intracellular alkalinization and acidification produced by NH4Cl exposure and withdrawal produced membrane hyperpolarization and depolarization, respectively, as expected for pHi-induced changes in gK+. By contrast, in the presence of HCO3-, NH4Cl exposure and withdrawal produced a decrease in apparent buffering capacity and changes in membrane potential difference consistent with compensatory regulation of electrogenic Na+/HCO3- cotransport. Moreover, the rate of pHi and potential difference recovery was several-fold greater in the presence as compared with the absence of HCO3-. Finally, continuous exposure to 10% CO2 in the presence of HCO3- produced intracellular acidification, and the rate of pHi recovery from intracellular acidosis was inhibited by Ba2+, which blocks pHi-induced changes in gK+, and by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, which inhibits Na+/HCO3- cotransport. These findings suggest that in hepatocytes, changes in transmembrane electrical potential difference, mediated by pH-sensitive gK+, play a central role in regulation of pHi through effects on electrogenic Na+/HCO3- cotransport.
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1. Two types of after‐potentials in the stretch receptor neurone of crayfish are described. 2. A short‐duration after‐hyperpolarization associated with a single spike or a few spikes is diminished and reversed on applying hyperpolarizing currents. However, a much longer‐lasting post‐tetanic hyperpolarization (PTH) is enhanced by conditioning hyperpolarization; thus, no reversal potential can be obtained. 3. No changes in membrane conductance occur during PTH. 4. Reducing K concentration in the bathing fluid diminishes PTH, while it shifts the reversal potential of the short after‐potential toward greater negativity. 5. Replacement of Na with Li, or addition of 2,4‐dinitrophenol in the bathing fluid suppresses PTH in a reversible manner. 6. Electrophoretic injection of Na into the cell induces a long‐lasting hyperpolarization. 7. No change in K‐equilibrium potential, as indicated by the reversal point of the short after‐potential, is detected during PTH. 8. It is concluded that the short after‐potential is caused by a permeability increase for potassium ion, whereas PTH is produced by an electrogenic Na‐pump.
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Abstract In the presence of Ba ++ , an increase in the bath HCO at constant CO 2 (i.e., Variable bath pH) produced a hyperpolarization. The hyperpolarizing effect of adding HCO 3 − /CO 2 at constant bath pH was not significantly affected by the presence of 50 μmol/l strophanthidin. In the absence of Ba ++ , addition of HCO 3 − /CO 2 at constant bath pH produced a Na + ‐dependent hyperpolarization. Therefore, CO 2 movements, electrogenic Na + /K + pump activity and changes in Ba ++ binding do not contribute significantly to the hyperpolarization induced by HCO 3 − . These results along with the results of previous studies (Astion et al: J Gen Physiol 93:731, 1989) strongly suggest that the hyperpolarization induced by the addition of HCO 3 − is due to an electrogenic Na + /HCO 3 − cotransporter, which transports Na + , HCO 3 − (or its equivalent), and net negative charge across the glial membrane. To study the role of electrogenic Na + /HCO cotransport in the regulation of pHi in glial cells, we used intracellular double‐barreled, pH‐sensitive microelectrodes. At a bath pH of 7.5, the mean initial intracellular pH (pH i ) was 7.32 (SD 0.03, n = 6) in HEPES‐buffered Ringer's solution and 7.39 (SD 0.1, n = 6) in HCO 3 − /CO 2 buffered solution. These values for pH i are more than 1.2 pH units alkaline to the pH i predicted from a passive distribution of protons; thus, these cells actively regulated pH i . Superfusion and with‐drawal of 15 mmol/l NH 4 + induced an acidification of 0.2 to 0.3 pH units, which recovered toward the original steady‐steady‐state pH i . Recovery from acidification was stimulated by adding HCO 3 − /CO 2 at constant pH. In HCO 3 − /CO 2 ‐buffered solutions, the recovery was Na + ‐dependent, inhibited by 4‐acetamido‐4′‐isothiocyanato‐stilbene‐2,2′‐disulfonic acid (SITS), and associated with a hyperpolarization of the membrane. Thus it appears that the electrogenic Na + /HCO 3 − cotransporter helps maintain the relatively alkaline pH i of glial cells and also contributes to the ability of glial cells to buffer changes in pH in the neuronal microenvironment.
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The Na + -K + -Cl - cotransporters (NKCCs), which belong to the cation-Cl - cotransporter (CCC) family, are able to translocate [Formula: see text] across cell membranes. In this study, we have used the oocyte expression system to determine whether the K + -Cl - cotransporters (KCCs) can also transport [Formula: see text] and whether they play a role in pH regulation. Our results demonstrate that all of the CCCs examined (NKCC1, NKCC2, KCC1, KCC3, and KCC4) can promote [Formula: see text] translocation, presumably through binding of the ion at the K + site. Moreover, kinetic studies for both NKCCs and KCCs suggest that [Formula: see text] is an excellent surrogate of Rb + or K + and that [Formula: see text] transport and cellular acidification resulting from CCC activity are relevant physiologically. In this study, we have also found that CCCs are strongly and differentially affected by changes in intracellular pH (independently of intracellular [[Formula: see text]]). Indeed, NKCC2, KCC1, KCC 2 , and KCC3 are inhibited at intracellular pH <7.5, whereas KCC4 is activated. These results indicate that certain CCC isoforms may be specialized to operate in acidic environments. CCC-mediated [Formula: see text] transport could bear great physiological implication given the ubiquitous distribution of these carriers.
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Sodium bicarbonate cotransport was studied in freshly dissociated Müller cells of the salamander retina. Variations in intracellular and extracellular pH evoked extracellular potassium concentration ([K+]o were recorded. Intracellular pH was measured by standard ratio imaging of the pH-sensitive dye BCECF, whereas extracellular pH was monitored by imaging BCECF fixed to coverslips under dissociated cells. Increasing [K+]o from 2.5 to 50 mM resulted in an intracellular alkalinization. The rate of alkalinization, 0.047 pH units/min, was reduced to 42% of control when HEPES was substituted for HCO3- and was reduced to 36% of control by the addition of 0.5 mM DIDS, a Na+/HCO3- cotransport blocker. The K(+)-evoked alkalinization was Cl(-)-independent and was not substantially reduced by amiloride or bumetanide. Increasing [K+]o to 50 mM also produced a rapid extracellular acidification, 0.01 to 0.05 pH units in amplitude. HEPES substitution and addition of 0.5 mM DIDS reduced the acidification to 7-8% of control, respectively. These results confirm the presence of a Na+/HCO3- cotransport system in salamander Müller cells and provide definitive evidence that glial cells can generate an extracellular acidification when [K+]o is increased. The K(+)-evoked extracellular acidification measured beneath cell endfeet was 304% of the amplitude of the acidification beneath cell somata, confirming that cotransporter sites are preferentially localized to the endfoot. The carbonic anhydrase inhibitor benzolamide (2 x 10(-5) M), which is poorly membrane permeant, increased the K(+)-evoked extracellular acidification to 269% of control, demonstrating that salamander Müller cells possess extracellular carbonic anhydrase.
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A slow hyperpolarization‐activated inwardly rectifying K + current ( I K(SHA) ) with novel characteristics was identified from the mouse embryonic hippocampus x neuroblastoma cell line HN9.10e. The non‐inactivating current activated negative to a membrane potential of −80 mV with slow and complex activation kinetics ( τ act ≈ 1–7 s) and a characteristic delay of 1–10 s (−80 to −140 mV) that was linearly dependent on the membrane potential. Tail currents and instantaneous open channel currents determined through fast voltage ramps reversed at the K + equilibrium potential ( E K ) indicating that primarily K + , but not Na + , permeated the channels. I K(SHA) was unaffected by altering the intracellular Ca 2+ concentration between ∼0 and 10 μ m , but was susceptible to block by 5 m m extracellular Ca 2+ , Ba 2+ ( K 1 = 0.42 m m ), and Cs + ( K 1 = 2.77 m m ) In cells stably transformed with M2 muscarinic receptors, I K(SHA) was rapidly, but reversibly, suppressed by application of micromolar concentrations of muscarine. At the single channel level K(SHA) channel openings were observed with the characteristic delay upon membrane hyperpolarization. Analysis of unitary currents revealed an inwardly rectifying I–V profile and a channel slope conductance of 7 pS. Channel activity persisted in the inside‐out configuration for many minutes. It is concluded that I K(SHA) m HN9.10e cells represents a novel K + current, which is activated upon membrane hyperpolarization. It is functionally different from both classic inwardly rectifying I kir currents and other cationic hyperpolarization‐activated 7 H currents that have been previously described in neuronal or glial cells.
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