Role of rafts in neurological disorders.

INTRODUCTION: Rafts are protein-lipid structural nanodomains involved in efficient signal transduction and the modulation of physiological processes of the cell plasma membrane. Raft disruption in the nervous system has been associated with a wide range of disorders. DEVELOPMENT: We review the concept of rafts, the nervous system processes in which they are involved, and their role in diseases such as Parkinson's disease, Alzheimer disease, and Huntington disease. CONCLUSIONS: Based on the available evidence, preservation and/or reconstitution of rafts is a promising treatment strategy for a wide range of neurological disorders.

Role of rafts in neurological disorders. / Participación de rafts en enfermedades neurológicas.

INTRODUCTION: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. DEVELOPMENT: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. CONCLUSIONS: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.

Anionic selectivity sequence of the Cl(-)-H+ symporter in the synaptosomal preparation from rat brain cortex.

The Na(+)/H(+) exchanger has been the only unequivocally demonstrated H(+)-transport mechanism in the synaptosomal preparation. We had previously suggested that a Cl(-)-H(+) symporter (in its acidifying mode) is involved in cytosolic pH regulation in the synaptosomal preparation. Supporting this suggestion, we now show that: (1) when synaptosomes are transferred from PSS to either gluconate or sulfate solutions, the Fura-2 ratio remains stable instead of increasing as it does in 50 mM K solution. This indicates that these anions do not promote a plasma membrane depolarization. (2) Based in the recovery rate from the cytosolic alkalinization, the anionic selectivity of the Cl(-)-H(+) symporter is NO(3)(-) > Br(-) > Cl(-) >> I(-) = isethionate = sulfate = methanesulfonate = gluconate. (3) PCMB 10 muM inhibits the gluconate-dependent alkalinization by 30 +/- 6%. (4) Neither Niflumic acid, 9AC, Bumetanide nor CCCP inhibits the recovery from the cytosolic alkalinization.

Participación de rafts en enfermedades neurológicas / Role of rafts in neurological disorders

Introducción: Los rafts constituyen nano-dominios estructurales de naturaleza lipoproteica que propician la eficiente transducción de señales y la modulación de procesos fisiológicos asociados a la membrana plasmática. En el sistema nervioso, la alteración de estos dominios se ha asociado con el desarrollo de diversos padecimientos. Desarrollo: En el presente artículo se revisa el concepto de rafts, los procesos del sistema nervioso en los cuales están involucrados y su papel en distintas afectaciones, entre las que se destacan las enfermedades de Parkinson, Alzheimer y Huntington. Conclusiones: Dadas las evidencias de su participación en diversas neuropatologías, la preservación y/o reconstitución de los rafts se vislumbran como una atractiva estrategia terapéutica.(AU)

Introduction: Rafts are function-structural cell membrane nano-domains. They contribute to explain the efficiency of signal transduction at the low physiological membrane concentrations of the signaling partners by their clustering inside specialized signaling domains. Development: In this article, we review the current model of the membrane rafts and their physio-pathological relevance in the nervous system, including their role in Parkinson, Alzheimer, and Huntington diseases. Conclusions: Rafts disruption/dysfunction has been shown to relate diverse neurological diseases. Therefore, it has been suggested that preservation of membrane rafts may represent a strategy to prevent or delay neuronal dysfunctions in several diseases.(AU)

Kainate, N-methylaspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro.

Kainate (0.62-5 mM) was found to increase the initial rate of influx of 45Ca and of 22Na into the non-inulin space of rat thin brain cortex slices incubated in vitro, and to shorten the equilibration time for both these ions. N-methyl-DL-aspartate (50-1000 microM), L-glutamate (0.62-5 mM), DL-homocysteate (0.62-2.5 mM), and ibotenate (6-170 microM) also significantly increased the influx of 45Ca into the non-inulin space of this preparation, while the non-neurotoxic acidic amino acids N-acetyl-L-aspartate, and alpha-methyl-DL-aspartate (both 1.25-5 mM), did not increase such influx. We suggest that enhanced calcium uptake may represent the basis for the neurotoxic effects of these compounds.

[Ca2+]i changes in guinea pig tracheal smooth muscle cells in culture: effects of Na+ and ouabain.

The objective of this work was to confirm that the contractile effects of ouabain and Na(+)-free solutions in guinea pig tracheal rings are associated with increments in the cytosolic free Ca2+ concentration ([Ca2+]i) in cultured tracheal smooth muscle (TSM) cells. Cultured cells were alpha-actin positive. Histamine (50 microM) and Na(+)-free solution elicited a transient increase in [Ca2+]i, while the responses to thapsigargin (1 microM) and ouabain (1 mM) were long lasting. However, carbachol (10, 200, and 500 mM) and high K(+)-solution produced no effect on [Ca2+]i, suggesting that cultured guinea pig TSM cells display a phenotype change but maintain some of the tracheal rings physiological properties. The transient rise in [Ca2+]i in response to the absence of extracellular Na+ and the effect of ouabain may indicate the participation of the Na(+)/Ca2+ exchanger (NCX) in the regulation of [Ca2+]i.

Parallel processing and selection of the responses to serotonin during reinnervation of an identified leech neuron.

In an attempt to define the mechanism of synaptic specificity, we have been studying pairs of identified leech neurons isolated in tissue culture. The cultured neurons reform specific synapses when paired with appropriate partners in the absence of other cell types. In recent studies, we have examined in detail the reformation of a serotoninergic synapse between the Retzius cell and one of its targets, the pressure sensitive (P) cell. The P cell in vivo and its soma in vitro have two types of responses to serotonin (5-HT). From voltage clamp analysis of cultured P cells, we demonstrated the parallel activation of chloride (gCls) and monovalent cation (gCations) channels coupled to distinct receptor subtypes and gated by separate second messengers. Only gCls was activated by 5-HT released from the presynaptic Retzius cell both in vivo and in vitro. This demonstrates the remarkable specificity of the reformation of this synapse in culture since only the correct 5-HT receptor subtype is activated. An 80% reduction of gCations was observed in P cells that had failed to be innervated by Retzius cells in culture, suggesting that gCations may be lost prior to synapse formation. Retzius cells depleted of 5-HT also reduced gCations in the paired P cells and incubating single P cells in 5-HT did not reduce gCations. In addition, aldehyde-fixed Retzius cells were able to selectively reduce gCations when paired with P cells. We conclude that the loss of gCations was due to contact between the neurons. The early clearing of counter-effective receptor subtypes may be a prelude to synapse formation.

Co-operative action of calcium ions in dopamine release from rat brain synaptosomes.

1. The release of [3H]dopamine from isolated presynaptic nerve terminals (synaptosomes) prepared from rat striata was measured as a function of the external Ca2+ concentration ([Ca2+]o). 2. In synaptosomes depolarized by the addition of 50 mM-K+, release of [3H]dopamine increased in a highly non-linear manner with [Ca2+]o. The release could be described as a third power function of [Ca2+]o. 3. Both 45Ca2+ influx and the change in the free cytosolic Ca2+ concentration ([Ca2+]i, measured with the fluorescent Ca2+ indicator fura-2) that were evoked by depolarization increased in a linear manner with [Ca2+]o. 4. These results suggest that non-linearity in the [Ca2+]o dependence of transmitter release originates in a co-operative relation between [Ca2+]i and exocytosis.

Selection of postsynaptic serotonin receptors during reinnervation of an identified leech neuron in culture.

Serotoninergic Retzius neurons reform an inhibitory synapse onto pressure-sensitive mechanosensory (P) neurons when the cells are removed from the nervous system of the leech and are juxtaposed in tissue culture. The somas of P cells in situ and single (uninnervated) P cells in culture have both a depolarizing and Cl-dependent hyperpolarizing response to application of the transmitter serotonin (5-HT). Synaptic release of 5-HT by a Retzius cell in situ and in culture evokes a Cl-dependent postsynaptic response but does not appear to activate the depolarizing receptors. We have characterized the ionic currents induced by synaptically released and applied 5-HT in voltage-clamped P cells in culture in order to determine the responses to transmitter and their modifications following innervation. When 5-HT was applied to single P cells, 2 types of channels were activated by 5-HT and could be distinguished by changing the ionic composition of the superfusion solution. In an impermeant cation (TrisCl) solution, a 5-HT-dependent Cl current was activated. When single P cells were superfused with a Cl-free solution (Cl replaced by impermeant SO4), 5-HT activated a monovalent cation current. Following innervation of a P cell by a cocultured Retzius cell, the reversal potential of the peak postsynaptic current depended on the Cl gradient and the synaptic response was blocked by the Cl channel blocker 9-anthracenecarboxylic acid. Thus, synaptic release of 5-HT activated solely the Cl channels and not the cationic channels. Pipette application of 5-HT onto innervated P cells activated a Cl conductance comparable in magnitude to the synaptic response. In contrast, the cationic conductance was reduced roughly 5-fold. It is concluded that innervation of a P cell by a Retzius cell resulted in clustering of the synaptic 5-HT receptors, which activate Cl channels and reduction of the nonsynaptic, cationic response. The result is a selection of receptors in the cultured P cell that mimics the pattern observed in vivo.

Role of sodium-calcium exchange in regulation of intracellular calcium in nerve terminals.

Ca efflux from rat brain presynaptic nerve terminals (synaptosomes) was examined after loading the terminals with 45Ca during a brief depolarization, usually in media containing 20 microM Ca labeled with 45Ca, to assure a small (physiological) load. Efflux of 45Ca was very slow in the absence of external Na and Ca (approximately 0.5% of the load/s) and was greatly accelerated by Na and/or Ca (presumably Na+-Ca2+ and Ca2+-Ca2+ exchange, respectively). The dependence of 45Ca efflux on external Na was sigmoid, with a Hill coefficient of approximately 2.5; this implies that more than two external Na ions are required to activate the efflux of one Ca ion. The external Na (Nao)-dependent Ca efflux was inhibited by 1 mM external La, by low temperature (Q10 congruent to 2.3), and by raising external K (to depolarize the synaptosomes). With small Ca loads, the mitochondrial uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), had negligible effect on either Ca uptake or efflux; with large loads (greater than or equal to 5 nmol/mg protein), however, FCCP reduced the depolarization-stimulated Ca uptake and increased the Nao-dependent Ca efflux. These effects may be attributed to reduction of mitochondrial Ca sequestration. Mitochondria do not appear to sequester much Ca when the loads are smaller (and more physiological). Estimations of Ca efflux indicate that approximately 20% of a small 45Ca load (approximately 0.75 nmol Ca/mg protein) may be extruded via Na+-Ca2+ exchange within 1 s; this corresponds to a net Ca efflux of approximately 110 pmol Ca X mg protein-1 X s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of cytosolic calcium in rat brain synaptosomes by sodium-dependent calcium efflux.

1. When pinched-off presynaptic nerve endings (synaptosomes) isolated from rat brain are incubated in a low-Na (24-36 mM) medium, they take up 45Ca in a time-dependent manner. In a medium containing 1 mM-Ca, this Na-dependent 45Ca uptake amounts to approximately 10 nmol/mg protein at 1 min, and to approximately 40 nmol/mg protein at 20 min. The Na-dependent Ca uptake is not reduced when the synaptosomes are loaded with concentrations of quin 2 as high as 2 mM. 2. The increase in 45Ca uptake is paralleled by an increase in the free cytosolic Ca concentration [Ca]i, as monitored with the fluorescent Ca indicators quin 2 or fura 2. [Ca]i increases from the value of approximately 200 to approximately 500 nM within 3-5 min, and thereafter, remains at this elevated level. 3. When synaptosomes that have been loaded with 45Ca (for 1 min, in a low-Na medium) are diluted into an Na-containing medium, there is a rapid efflux of the Ca load. After correcting for Ca that is taken up during the efflux period, calculations show that the total Ca in the synaptosomes returns to the control level within 1 min. Measurements of total chemical Ca parallel the measurements made with radiotracer Ca, and confirm that the Ca loaded into the nerve terminals during a 5 min incubation in a low-Na medium is extruded from the nerve terminals within 1 min in a normal-Na medium. 4. The efflux of Ca from the synaptosomes is paralleled by a drop of [Ca]i to its basal level, also within 1 min. 5. The mitochondrial uncoupler, carbonyl cyanide p-trifluoromethyloxy-phenyl-hydrazone (FCCP, 1 microM), has no effect on either Na-dependent Ca uptake or efflux in synaptosomes. FCCP causes a slight (100-200 nM) increase in [Ca]i in synaptosomes resuspended in either a Na or a low-Na medium. This indicates that little of the Ca that is taken up by the synaptosomes in a low-Na medium is sequestered by the mitochondria. 6. These results suggest that Na-dependent Ca efflux (probably Na-Ca exchange) plays an important role in allowing nerve terminals to recover rapidly from a Ca load.

Distinct receptors, second messengers and conductances underlying the dual responses to serotonin in an identified leech neurone.

1. Pressure-sensitive mechanosensory (P) neurones of the leech Hirudo medicinalis produce two responses to serotonin (5-HT): activation of a Cl- conductance and of a non-selective monovalent cation conductance. The effects of channel blockers, the receptor pharmacology and the second-messenger dependence of these responses were studied in voltage-clamped P cells in culture. Antagonists were applied by superfusion and agonists by pressure ejection. 2. Zn2+ (100 mumol l-1) and H+ (pH 6.5 and lower) reversibly reduced the Cl- conductance activated by 5-HT. The cation conductance was impermeant to calcium ions and was reduced by micromolar concentrations of the Na+ channel inhibitors amiloride and 3,4-dichlorobenzamil. 3. High concentrations of antagonists or agonists of 5-HT1 receptors and an antagonist of 5-HT3 receptors had no effect on either response of P cells to 5-HT. Micromolar concentrations of ketanserin or cyproheptadine, which selectively antagonize 5-HT2 receptors, reduced the cation but not the Cl- conductance. From these results, the receptor underlying the cation conductance appears to be of the 5-HT2 subtype, whereas the receptor activating the Cl- conductance does not fit within the mammalian classification scheme. 4. Brief (less than 500 ms) application of membrane-permeant agonists of the second messenger cyclic AMP elicited a Cl- conductance, whereas antagonists of cyclic-AMP-dependent protein kinase A reversibly suppressed the Cl- conductance elicited by 5-HT and by cyclic AMP agonists. Compounds affecting other second messenger pathways were without effect on the Cl- conductance. It therefore appears that the Cl- conductance is activated by cyclic-AMP-dependent protein kinase A. 5. Cyclic nucleotide agonists and antagonists were without effect on the cation conductance. However, brief application of phorbol esters, which activate protein kinase C, elicited an amiloride-sensitive cation current. An inhibitor of protein kinase C reduced the cation conductance activated by 5-HT and by phorbol esters. Therefore, the cation conductance appears to depend on activation of protein kinase C. 6. We conclude that 5-HT activates two types of receptor coupled to separate ionic channels via different second messenger pathways in P cells. A receptor that is distinct from the mammalian subtypes activates Cl- channels via cyclic-AMP-dependent protein kinase A. 5-HT2 receptors appear to activate cation channels by means of protein kinase C.

Regulation of pH in rat brain synaptosomes. II. Role of Cl-.

1. We have previously shown that rat brain synaptosomes exhibit a very dynamic Na+/H+ exchanger. We have also observed that although synaptosomes lack HCO3(-)-based transport mechanisms, they do respond with changes in pHi upon Clo- removal. 2. Here we show that when synaptosomes are transferred from Ringer solution (RS) to Cl(-)-free RS, there is a cytosolic alkalinization of approximately 0.22 pH units. This phenomenon is DIDS (4,4'-diisothiocyanostilbene-3,3'-disulfonic acid) inhibitable. The alkalinization is completely reversed when Cl- ions are reintroduced. The presence of HCO3- or Ca2+ does not modify the response to Cl(-)-removal or replenishment. 3. In acid-loading experiments, the initial rate of pHi recovery is higher in Cl(-)-free RS than in RS. The final resting pHi after the recovery in Cl(-)-free RS is approximately 0.22 pH units higher than that obtained in media containing Cl-. The magnitude of the NaOAc-induced acidification is 2.5-fold larger in the presence than in the absence of Cl-. Similar results are obtained in the presence of HCO3-. 4. These data suggest that H+ movements may be coupled to Cl- movements. To study this possibility further, we developed a technique to simultaneously measure H+ and Cl- by using the fluorescence of 5' (and 6')-carboxy-10-dimethylamino-3-hydroxy-spyro-[7H benzo[c]xanthene- 7,1'(3'H)-isobenzofuran]3'-one (SNARF-1) and MQAE [N-(6-methoxyquinolyl)acetoxy ester], respectively. 5. Our results indicated that the steady-state [Cl-]i in synaptosomes is approximately 56 mM, thus indicating that Cli- is not passively distributed.(ABSTRACT TRUNCATED AT 250 WORDS)

Contact-mediated loss of the nonsynaptic response to transmitter during reinnervation of an identified leech neuron in culture.

We have examined the modification of responses to transmitter during the reformation of a specific synapse by identified leech neurons in culture. Single pressure-sensitive mechanosensory (P) neurons in culture and the soma of this cell in vivo have 2 conductances that are activated by application of 5-HT: a Cl conductance and a monovalent cation conductance (gCations); synaptic release of 5-HT by a serotoninergic Retzius cell in vivo and in culture activates only the Cl conductance and not gCations in the P cell. We have characterized the loss of gCations in P cells by manipulating the culture conditions. When 5-HT was applied from a pipette, innervated P cells and P cells paired with Retzius cells that had not formed synapses had a gCations that was markedly reduced compared with gCations in the single P cell. When 5-HT was included in the culture medium in which single P cells were grown, gCations was not reduced. When Retzius-P cell pairs were treated with reserpine, which was shown to deplete the presynaptic neuron of 5-HT, gCations in the P cell was as low as in untreated pairs. Pairing aldehyde-fixed Retzius cells with untreated P cells also resulted in the loss of gCations. The Retzius cell had both types of receptors, but synapse formation did not affect gCations in the presynaptic neuron. The results demonstrate that the loss of the nonsynaptic response of the P cell to transmitter is due to contact between the neurons rather than to the release of transmitter or trophic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Molecular mechanism of noradrenaline transport by rat auricles in vitro]. / Mecanismos moleculares del transporte de noradrenalina por auriculas de rata in vitro

The transport of tritium-labelled noradrenaline (3H-NA) into bisected rat auricular appendages in vitro, has been studied, using 14C-inulin as an extracellular space tracer, thus allowing a precise measurement of the 3H-NA transported into the cells. The NA transport time course is relatively fast, and initial rates lasted 5 min. in 150 mM sodium, 3 min. in 26 mM sodium, and about 1 min. in 50 mM potassium. Intracellular accumulation of 3H-NA by synaptic vesicles, was found not to be important in the first minute of transport. In the presence of 150 mM sodium a transport Km for NA of 0.59 +/- 0.06 muM (mean +/- S.E.M.) and a maximal velocity (Vmax.) of 2.44 +/- 0.43 pmol/mg. protein/min. were estimated. When sodium was lowered to 26 mM, the Km increased to 2.26 +/- 0.7 muM (P less than 0.001), while Vmax. showed no change. With 0 mM sodium (choline substitution) active NA transport is completely suppressed, and only a diffusional component can be discerned. No binding of NA to beta adrenergic receptors was found, and a small but highly significant binding to the non-specific catechol receptors could be detected.

Strontium, barium, and manganese metabolism in isolated presynaptic nerve terminals.

To gain insight into the mechanisms by which the divalent cations Sr, Ba, and Mn affect neurotransmitter release from presynaptic nerve terminals, we examined the sequestration of these cations, in comparison to Ca, by mitochondrial and nonmitochondrial [presumably smooth endoplasmic reticulum (SER)] organelles and the extrusion of these cations from isolated nerve terminals. Sequestration was studied in synaptosomes made leaky to small ions by treatment with saponin; efflux was examined in intact synaptosomes that were preloaded with the divalent cations by incubation in depolarizing (K rich) media. The selectivity sequence for ATP-dependent mitochondrial uptake that we observed was Mn much greater than Ca greater than Sr much greater than Ba, whereas that for the SER was Ca greater than or equal to Mn greater than Sr much greater than Ba. When synaptosomes that were preloaded with divalent cations were incubated in Na- and Ca-free media, there was little efflux of Ca, Ba, Sr, or Mn. When the incubation was carried out in media containing Na without Ca, there was substantial stimulation of Ca and Sr efflux, but only slight stimulation of Ba or Mn efflux. In Na-free media, the addition of 1 mM Ca promoted the efflux of all four divalent cations, probably via Ca-divalent cation exchange. In summary, the sequestration and extrusion data suggest that, with equal loads, Mn will be buffered to the greatest extent, whereas Ba will be least well buffered. These results may help to explain why Mn has a very long-lasting effect on transmitter release, while the effect of Sr is much briefer.

Abnormalities in CD69 expression, cytosolic pH and Ca2+ during activation of lymphocytes from patients with systemic lupus erythematosus.

Several immuno-regulatory abnormalities have been described in SLE patients. T cell dysfunction in SLE includes defective in vitro proliferative responses to several stimuli, reduced IL-2 production and a poor helper function. It has been widely proposed that this defective T cell immunoregulatory function has a key role in the hyperactivity of B cells and auto-antibody production in SLE. However, it has not been elucidated whether or not this cell dysfunction is intrinsic to lymphocytes or is due to other factors such as anti-lymphocyte auto-antibodies. In this study we have evaluated some important early cell activation events in T and non-T lymphocytes from patients with systemic lupus erythematosus (SLE). Peripheral blood lymphocytes from SLE patients and controls were isolated. The intracellular pH (pHi), cytosolic calcium (Ca2+i) and CD69 expression were determined by spectrofluorometry and flow cytometry. Modifications of these parameters in response to protein kinase C (PKC) activators, mitogenic lectins and calcium ionophores were also studied. We found a significant reduction in the increase of pHi in response to PKC activators (PMA) in SLE cells. In addition, the induction of CD69 expression by PMA was significantly lower in T cells from SLE patients. By contrast, freshly isolated non-stimulated SLE cells exhibited a significantly higher pHi, as well as an increased baseline expression of the early cell activation antigen CD69. On the other hand, the increase in Ca2+i in response to a Ca2+ ionophore (4Br-A23187) or thapsigargin in Ca(2+)-free solutions, was smaller in SLE lymphocytes. We concluded that T cells from SLE patients exhibit abnormalities in several key early cell activation events (pHi, Ca2+i and CD69 expression). These abnormalities could have an important role in the T cell dysfunction observed in SLE. The presence of T cells with a preactivated phenotype in the peripheral blood of SLE patients, could be a reflection of the ongoing autoimmune phenomena that is occurring in these patients.

Regulation of pH in rat brain synaptosomes. I. Role of sodium, bicarbonate, and potassium.

1. We investigated the regulation of intracellular pH (pHi) in rat brain isolated nerve terminals (synaptosomes), using fluorescence pH indicators and time-resolved fluorescence spectroscopy. 2. The resting pHi was not significantly affected by the presence or absence of HCO3-. Removal of external Na+, in the absence or presence of HCO3- caused a rapid acidification of pHi. The recovery from acid loads was primarily due to the activity of the Na+/H+ exchanger, confirming the relevance of this transport system in synaptosomes. 3. Our data revealed that in synaptosomes the activity of the Na+/H+ exchanger was not regulated by either protein kinase C or kinase A. In contrast, Ca2+ played an important role in the regulation of Na+/H+ exchanger. This was supported by the observation that 4Br-A23187 induced a Na(+)-dependent alkalinization of the resting pHi and greatly enhanced the initial rate and the degree of the recovery from acid loads. 4. In most eukaryotic cells, HCO3(-)-based transport mechanisms play an important role in pHi regulation. In synaptosomes, however, HCO3- transport is not significantly involved in pHi regulation, because the presence or absence of HCO3- does not affect resting pHi nor the rate of pHi recovery to acid loads. Further studies to address the role of Cl- and HCO3- in pHi regulation in synaptosomes are discussed in the companion paper. 5. Increasing the concentration of Ko+ also resulted in a rise of steady-state pHi by a processes that is Ca2+ and HCO3- independent. This alkalinization could be due to either K+/H+ exchanger activity, K(+)-induced depolarization, reduction of delta microH+, or a direct reduction of delta microK+. Calculated H+ driving forces suggest that the reduction in the inwardly directed H+ leak is sufficient to explain this K(+)-induced alkalinization because it changes the delta microH+ by virtue of setting the membrane potential difference (Em) to the K+ equilibrium potential (EK+).