Intracellular Alkalization Research Articles

Lidocaine depolarizes the mitochondrial membrane potential by intracellular alkalization in rat dorsal root ganglion neurons

The mitochondrial membrane potential (ΔΨm) is an important factor for apoptosis, and it is produced by the proton electrochemical gradient (ΔµH(+)). Therefore, the intracellular proton concentration (pH(in)) is an important factor for modifying the ΔΨm. However, the effects of lidocaine on pH(in) are unclear. To investigate mitochondrial responses to lidocaine, therefore, we simultaneously measured pH(in) with ΔΨm, flavin adenine dinucleotide (FAD), and reduced form of nicotinamide adenine dinucleotide (NADH) fluorescence, and calculated the FAD/NADH ratio (redox ratio), the superoxide production in mitochondria. Morphological change and early apoptosis were observed by annexin-V FITC staining under fluorescent microscope. The ratiometric fluorescent probe JC-1 and HPTS were used for the simultaneous measurements of ΔΨm with pH(in) in rat dorsal root ganglion (DRG) neurons. FAD and NADH autofluorescence were simultaneously measured, and the FAD/NADH fluorescence ratio (redox ratio) was calculated. The superoxide was measured by mitosox-red fluorescent probe for mitochondrial superoxide. Lidocaine was evaluated at 1, 5, and 10mM. Morphological change and early apoptosis were observed after 10mM lidocaine administration. Lidocaine depolarized ΔΨm with increased pH(in) in a dose-dependent manner. In low-pH saline (pH 6), in the presence of both the weak acids (acetate and propionate), lidocaine failed to depolarize ΔΨm and increase pH(in). On the other hand, lidocaine decreased the redox ratio in the cell and increased the levels of superoxide in a dose-dependent manner. These results demonstrated that lidocaine depolarizes ΔΨm by intracellular alkalization. These results may indicate one of the mechanisms responsible for lidocaine-induced neurotoxicity.

Effect of aldosterone on the amplification of oncolytic vaccinia virus in human cancer lines

Background/AimsJX-594 is an oncolytic virus derived from the Wyeth vaccinia strain that causes replication-dependent cytolysis and antitumor immunity. Starting with a cross-examination of clinical-trial samples from advanced hepatocellular carcinoma patients having high levels of aldosterone and virus amplification in JX-594 treatment, we investigated the association between virus amplification and aldosterone in human cancer cell lines.MethodsCell proliferation was determined by a cell-counting-kit-based colorimetric assay, and vaccinia virus quantitation was performed by quantitative polymerase chain reaction (qPCR) and a viral plaque assay. Also, the intracellular pH was measured using a pH-sensitive dye.ResultsSimultaneous treatment with JX-594 and aldosterone significantly increased viral replication in A2780, PC-3, and HepG2 cell lines, but not in U2OS cell lines. Furthermore, the aldosterone treatment time altered the JX-594 replication according to the cell line. The JX-594 replication peaked after 48 and 24 hours of treatment in PC-3 and HepG2 cells, respectively. qPCR showed that JX-594 entry across the plasma membrane was increased, however, the changes are not significant by the treatment. This was inhibited by treatment with spironolactone (an aldosterone-receptor inhibitor). JX-594 entry was significantly decreased by treatment with EIPA [5-(N-ethyl-N-isopropyl)amiloride; a Na+/H+-exchange inhibitor], but aldosterone significantly restored JX-594 entry even in the presence of EIPA. Intracellular alkalization was observed after aldosterone treatment but was acidified by EIPA treatment.ConclusionsAldosterone stimulates JX-594 amplification via increased virus entry by affecting the H+ gradient.

Angiotensin II inhibits the electrogenic Na+/HCO3− cotransport of cat cardiac myocytes

The Na+/HCO3− cotransporter (NBC) plays an important role in intracellular pH (pHi) regulation in the heart. In the myocardium co-exist the electrogenic (eNBC) and electroneutral (nNBC) isoforms of NBC. We have recently reported that angiotensin II (Ang II) stimulated total NBC activity during the recovery from intracellular acidosis through a reactive oxygen species (ROS) and ERK-dependent pathway. In the present work we focus our attention on eNBC. In order to study the activity of the eNBC in isolation, we induced a membrane potential depolarization by increasing extracellular K+ [K+]o from 4.5 to 45mM (K+ pulse). This experimental protocol enhanced eNBC driving force leading to intracellular alkalization (0.19±0.008, n=6; data expressed as an increase of pHi units after 14min of applying the K+ pulse). This alkalization was completely abrogated by the NBC blocker S0859 (−0.004±0.016*, n=5; * indicates p<0.05 vs control) but not by the Na+/H+ exchanger blocker HOE642 (0.185±0.04, n=4), indicating that we are exclusively measuring eNBC. The K+ pulse induced alkalization was canceled by 100nM Ang II (−0.008±0.018*; n=5). This inhibitory effect was prevented when the myocytes were incubated with losartan (AT1 receptor blocker, 0.18±0.02; n=4) or SB202190 (p38 MAP kinase inhibitor, 0.25±0.06; n=5). Neither chelerythrine (PKC inhibitor, −0.06±0.04*; n=4), nor U0126 (ERK inhibitor, −0.07±0.04*; n=4) nor MPG (ROS scavenger, −0.02±0.05*; n=8) affected the Ang II-induced inhibition of eNBC. The inhibitory action of Ang II on eNBC was corroborated with perforated patch-clamp experiments, since no impact of the current produced by eNBC on action potential repolarization was observed in the presence of Ang II. In conclusion, we propose that Ang II, binding to AT1 receptors, exerts an inhibitory effect on eNBC activity in a p38 kinase-dependent manner.

Local Anesthetics Depolarize Mitochondrial Membrane Potential by Intracellular Alkalization in Rat Dorsal Root Ganglion Neurons

Although it has been reported that local anesthetics, especially lidocaine, are cytotoxic, the mechanism is unclear. Depolarization of the mitochondrial membrane potential (DeltaPsim), one of the markers of mitochondrial failure, is regulated by the proton electrochemical gradient (Delta H(+)). Therefore, intracellular pH ([pH]in) and mitochondrial pH ([pH]m) are important factors for modifying DeltaPsim. However, the effects of local anesthetics on [pH]in and [pH]m are unclear. To investigate mitochondrial responses to local anesthetics, we simultaneously measured [pH]m and [pH]in, along with DeltaPsim. The ratiometric fluorescent probe JC-1 and HPTS were used for the simultaneous measurements of DeltaPsim with [pH]in in rat dorsal root ganglion neurons. A carboxy-SNARF-1 fluorescent probe was used to measure [pH]m. Lidocaine, mepivacaine, bupivacaine, procaine, QX-314, a charged form of lidocaine, and ammonium chloride (NH(4)Cl) were evaluated. DeltaPsim was depolarized and [pH]in was increased by lidocaine, mepivacaine, bupivacaine, and procaine in a dose-dependent manner. Significantly, a relationship between DeltaPsim and [pH]in was observed for lidocaine, mepivacaine, bupivacaine, procaine, and NH(4)Cl perfusion. In contrast, QX-314 did not change DeltaPsim or [pH]in. In low-pH saline (pH6) and in the presence of a weak acid, lidocaine failed to increase [pH]in or depolarize DeltaPsim. The [pH]m was also increased by lidocaine, mepivacaine, bupivacaine, procaine, and NH(4)Cl. These results demonstrate that uncharged (base) forms of local anesthetics induce DeltaPsim depolarization. One of the causes is intracellular and mitochondrial alkalization.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

Activation and Regulation of Nociceptive Transient Receptor Potential (TRP) Channels, TRPV1 and TRPA1

TRP channels are well recognized for their contributions to sensory transduction, responding to a wide variety of stimuli including temperature, nociceptive stimuli, touch, osmolarity and pheromones. In particular, the involvement of TRP channels in nociception has been extensively studied following the cloning of the capsaicin receptor, TRPV1. Painful diabetic peripheral neuropathy is described as a superficial burning pain, and it is one of the most commonly encountered neuropathic pain syndromes in clinical practice. We found that hypoxic and high glucose conditions commonly observed in diabetes potentiate TRPV1 activity without affecting TRPV1 expression both in native rat sensory neurons and HEK293 cells expressing rat TRPV1. The potentiation seems to be caused by phosphorylation of the serine residues of TRPV1 by PKC. These data indicate that PKC-dependent potentiation of TRPV1 activities under hypoxia and hyperglycemia might be involved in early diabetic neuropathy. Mechanisms for the detection of alkaline pH by sensory neurons are not well understood, although it is well accepted that acidic pH monitoring can be attributed to several ion channels, including TRPV1 and ASICs. We found that alkaline pH activates TRPA1 and that the TRPA1 activation is involved in nociception, using Ca(2+)-imaging and patch-clamp methods. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors, which were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1.

Influence of pH on the UV sensitivity of peritoneal macrophage plasma membrane

Lowering the extracellular pH (from 7.2 to 6.3) or intracellular acidification in isolated murine peritoneal macrophages before UV-irradiating them to 9 J/cm2 (λmax = 306 nm) diminish the percentage of cells with damaged membranes. Extracellular pH 8.4 or intracellular alkalization have an opposite effect. After transient hypoosmotic swelling, the UV-induced membrane damage is fully pronounced regardless of external pH. In cells that survive UV-irradiation to 8 and 10 J/cm2 (λmax = 297 nm), the intracellular pH is 0.2 and 0.25 unit lower than in nonirradiated cells.

Gating, permselectivity and pH‐dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to approximately 400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is approximately 90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pH(i) from 7.2 to 7.8 increased gap junctional conductance (g(j)) of approximately 100-fold with pK(a) = 7.41. We show that this g(j) increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca(2+)](o) = approximately 0.3 mm or below, demonstrating that a moderate reduction of [Ca(2+)](o) can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pH(i) with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell-cell coupling in horizontal cell in the retina.

The regulation of matrix metalloproteases by NHE1 in non‐small lung cancer cells

The Na+‐H+ exchanger isoform‐1 (NHE1) has plays a major role in the coordination of cell migration and cancer metastasis. One possible mechanism is based on a family of proteases, matrix metalloproteases (MMPs), which degrade the extracellular matrix allowing cells to escape tumors and migrate. Studying three non‐small cell lung carcinoma cell lines (NCI‐H358, ‐H460, ‐1299) we have demonstrated using zymogen gels that stimulation with urokinase plasminogen activator (uPA) leads to an increase in MMP9 activity. Additionally, when NHE1 is inhibited with ethylisopropyl amiloride (EIPA), MMP9 activity decreases. Here we investigated whether the increase in MMP9 activity was due to increased activation or increased expression of MMP9. We hypothesized that NHE1 drives an intracellular alkalization that promotes MMP‐9 expression. To determine this action, stimulated and EIPA inhibited cells were analyzed for the level of RNA transcription measured by qPCR. Expression of MMP9 in H460 and H358 show a 3‐4 fold increase in transcription while H1299 displays a 100 fold increase in MMP‐9 transcription when stimulated with uPA. MMP1 transcription displays a 14 and 66 fold increase in H460 and H358 under NHE1 inhibition, but the H1299 showed no increase in MMP‐1 with EIPA treatment. This work was supported by NIH grant 1R15CA135616‐01, and NSF grant MCB‐0817784.

TRPV1 Is Activated by Both Acidic and Basic pH

Maintaining physiological pH is required for survival, and exposure to alkaline chemicals such as ammonia (smelling salts) elicits severe pain and inflammation through unknown mechanisms. TRPV1, the capsaicin receptor, is an integrator of noxious stimuli including heat and extracellular acidic pH. Here, we report that ammonia activates TRPV1, TRPA1 (another polymodal nocisensor), and other unknown receptor(s) expressed in sensory neurons. Ammonia and intracellular alkalization activate TRPV1 through a mechanism that involves a cytoplasmic histidine residue, not used by other TRPV1 agonists such as heat, capsaicin or low pH. Our studies show that TRPV1 detects both acidic and basic deviations from homeostatic pH.

Intracellular alkalization causes pain sensation through activation of TRPA1 in mice

Vertebrate cells require a very narrow pH range for survival. Cells accordingly possess sensory and defense mechanisms for situations where the pH deviates from the viable range. Although the monitoring of acidic pH by sensory neurons has been attributed to several ion channels, including transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs), the mechanisms by which these cells detect alkaline pH are not well understood. Here, using Ca2+ imaging and patch-clamp recording, we showed that alkaline pH activated transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and that activation of this ion channel was involved in nociception. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration, indicating that alkaline pH activated TRPA1 from the inside. Analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1 and may provide a molecular explanation for some of the human alkaline pH-related sensory disorders whose mechanisms are largely unknown.

Ageladine A, a pyrrole–imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye

The alkaloid ageladine A, a pyrrole–imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue–green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.

Effect of Tris-Hydroxymethyl Aminomethane on intracellular pH depends on the extracellular non-bicarbonate buffering capacity

The effect of Tris-Hydroxymethyl Aminomethane (THAM) on intracellular pH (pHi) is unknown. We previously demonstrated that the effect of sodium bicarbonate on pHi depends on the non-bicarbonate buffering system. First, human hepatocytes from hepatocytes cell culture (HepG2) were perfused with an acidotic artificial medium containing 5-mmol/L (H5) or 30-mmol/L (H30) concentrations of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES), a non-bicarbonate buffer. We studied the effect of THAM on the pHi in both conditions. We repeated the same protocol using an acidotic human blood with a 5% or 40% hematocrit. The pHi was measured with the pH-sensitive fluorescent dye bis-carboxyethyl carboxy-fluorescein (BCECF). Gas analysis was performed before and during the alkaline infusion. The results showed that THAM caused an intracellular alkalization that was higher when the non-bicarbonate buffer concentration was low (0.45 +/- 0.21 and 0.22 +/- 0.14 pH units with H5 and H30, respectively). A significant relationship was found between changes in pHi and changes in PCO(2). Similar results were obtained with the human blood. In conclusion, the intracellular alkalizing effect of THAM is caused by the induced decrease of PCO(2) linked to the extracellular non-bicarbonate buffer capacity: The smaller the concentration of extracellular non-bicarbonate buffer, the higher the PCO(2) decrease caused by THAM.

Characterization of Na/H exchange systems in immortalized rat caput epididymal (RCE) cells

RCE cells are primary cultures of rat caput epididymal cells immortalized with SV40 large T antigens. The goals of the present studies were to characterize the endogenous Na/H exchange systems in these cells and to understand the regulation of endogenous Na/H exchangers by hyperosmolarity because epididymal fluids are hypertonic. By Western blotting, RCE cells were shown to have endogenous NHE1 (105kD protein), but not NHE2 and NHE3. For measurement of Na/H exchange with BCECF, RCE cells were grown on collagen coated glass cover slips. These cells exhibited Na dependent alkalinization which was completely inhibited by HOE694 (IC50 = 0.3μM), confirming the presence of NHE1. Kinetic analysis with Na showed a Km of 18±3mM with a Hill coefficient of 1.1±0.2. The acute effect of hyperosmolarity on NHE1 was studied at both steady-state pH and by initial rates of pH recovery after NH4Cl acidification. Addition of 100mM mannitol at steady-state pH of cells caused intracellular alkalization (ΔpH = 0.4±0.1) which was blocked by HOE694. Mannitol (100mM) also increased the initial pH recovery rates of cells. Kinetic analysis suggested that there was no change in Vmax but a change in Km(H) (Km(H): control; 6.51±0.01 vs mannitol; 6.75±0.01). Interestingly, mannitol stimulation of NHE1 was inhibited by BAPTA (50μM) and by the calmodulin (CaM) inhibitor, W13 (45μM), suggesting the involvement of Ca/CaM. The chronic effect of hyperosmolarity on NHE1 was also studied by culturing the cells in the presence of 100mM mannitol for 72h. Under this culturing condition, the amount of NHE1 protein was increased by 2.1 fold as determined by Western blotting. We conclude that RCE cells contain endogenous NHE1 which is stimulated by hyperosmolarity in a Ca/CaM dependent manner. Chronic exposure of RCE cells in hypertonic medium increases the amount of NHE1 protein.

Involvement of AE3 isoform of Na +-independent Cl −/HCO 3− exchanger in myocardial pH i recovery from intracellular alkalization

Myocardial pH i recovery from intracellular alkalization results in part from the acid load (− J H+) carried by Cl −/HCO 3 − anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH i homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH i regulation. We developed rabbit polyclonal antibodies against the extracellular “loops”: one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH i regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased − J H+ in response to myocardial alkalization (from a mean control value of 1.06 ± 0.26 to 0.32 ± 0.13 mmol/L/min, n = 7, P < 0.05) without affecting the baseline pH i (7.22 ± 0.03 vs. 7.21 ± 0.04). The anti-AE3 loop 4 antibody did not modify either pH i recovery or baseline pH i. Under control conditions, endothelin-1 (ET-1) increased − J H+ in response to myocardial alkalization from 1.30 ± 0.18 to 2.01 ± 0.33 mmol/L /min ( n = 5, P < 0.05). This effect of ET-1 on − J H+ was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH i recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH i.

Base Treatment Corrects Defects Due to Misfolding of Mutant Cystic Fibrosis Transmembrane Conductance Regulator

The most common form of the disease-causing cystic fibrosis transmembrane conductance regulator mutation, DeltaF508, leads to a misfolded protein that undergoes endoplasmic reticulum-associated degradation. Retrieval of misfolded protein from the cis-Golgi or pre-Golgi intermediate compartment is a critical factor in endoplasmic reticulum retention and degradation of DeltaF508 protein. Therefore, the inhibition of retrograde Golgi-to-endoplasmic reticulum traffic by the alkalinization of Golgi lumen may permit functional DeltaF508 protein to reach the cell surface. Functional and biochemical effects of alkaline treatment on misfolded cystic fibrosis transmembrane conductance regulator-induced defects were measured in CFPAC-1 cells, which endogenously express DeltaF508 cystic fibrosis transmembrane conductance regulator, and in CHO cells, which heterologously express DeltaF508 cystic fibrosis transmembrane conductance regulator. The animal survival rate and the functional expression of cystic fibrosis transmembrane conductance regulator proteins were analyzed in homozygous DeltaF508 mice after chronic treatment with weak base NaHCO3. In CFPAC-1 and CHO cells, intracellular alkalization by reducing carbon dioxide concentrations in a carbon dioxide incubation chamber or intra-Golgi alkalization by bafilomycin A1 treatment increased the membrane expression of DeltaF508 protein and cystic fibrosis transmembrane conductance regulator-dependent anion transport. Notably, chronic administration of NaHCO3 increased the long-term survival of homozygous DeltaF508 mice and induced the functional expression of cystic fibrosis transmembrane conductance regulator in the luminal membrane of intestinal epithelium. We found that base treatments correct misfolded cystic fibrosis transmembrane conductance regulator-induced defects in vitro and in vivo. These results imply that the alkalization of intracellular compartments, in particular, Golgi or pre-Golgi intermediate compartments, can be a potential therapeutic target for the loss-of-function type of conformational diseases.

Regulation of human airway ciliary beat frequency by intracellular pH.

pHi affects a number of cellular functions, but the influence of pHi on mammalian ciliary beat frequency (CBF) is not known. CBF and pHi of single human tracheobronchial epithelial cells in submerged culture were measured simultaneously using video microscopy (for CBF) and epifluorescence microscopy with the pH-sensitive dye BCECF. Baseline CBF and pHi values in bicarbonate-free medium were 7.2 +/- 0.2 Hz and 7.49 +/- 0.02, respectively (n = 63). Alkalization by ammonium pre-pulse to pHi 7.78 +/- 0.02 resulted in a 2.2 +/- 0.1 Hz CBF increase (P < 0.05). Following removal of NH4Cl, pHi decreased to 7.24 +/- 0.02 and CBF to 5.8 +/- 0.1 Hz (P < 0.05). Removal of extracellular CO2 to change pHi resulted in similar CBF changes. Pre-activation of cAMP-dependent protein kinase (10 microM forskolin), broad inhibition of protein kinases (100 microM H-7), inhibition of PKA (10 microM H-89), nor inhibition of phosphatases (10 microM cyclosporin + 1.5 microM okadaic acid) changed pHi-mediated changes in CBF, nor were they due to [Ca2+]i changes. CBF of basolaterally permeabilized human tracheobronchial cells, re-differentiated at the air-liquid interface, was 3.9 +/- 0.3, 5.7 +/- 0.4, 7.0 +/- 0.3 and 7.3 +/- 0.3 Hz at basolateral i.e., intracellular pH of 6.8, 7.2, 7.6 and 8.0, respectively (n = 18). Thus, intracellular alkalization stimulates, while intracellular acidification attenuates human airway CBF. Since phosphorylation and [Ca2+]i changes did not seem to mediate pHi-induced CBF changes, pHi may directly act on the ciliary motile machinery.

Blockers of carbonic anhydrase can cause increase of retinal capillary diameter, decrease of extracellular and increase of intracellular pH in rat retinal organ culture.

At least in normal-pressure glaucoma a vascular genesis with hypoperfusion and regulation impairment is discussed. This may lead to malnutrition of retinal ganglion cells and apoptosis. The retinal microvasculature has a small functional reserve. In addition, the retinal microvessels lack the autonomic nerves that are normally found in other tissues. Thus, no systemic influences reach the retinal capillaries apart from circulating hormones or transmitters. Blockers of carbonic anhydrase (CA) may modulate regional blood flow by mediating changes in extra- and intracellular pH. However, it is still unclear (1) whether blockers of CA really change the pH near the retinal capillaries and (2) how changes in the local pH affect the capillary tone in situ. Therefore, we tested dorzolamide and acetazolamide in our model of the freshly enucleated rat retina. Adult Sprague-Dawley rats (of both sexes, 250-350 g) were killed and retinae were prepared. The retinae were gently separated from the retinal pigmented epithelium and were observed in a chamber for electronic light microscopy or were fixed for immunohistochemistry. Electronic light microscopy of the retinal cells was performed with a Zeiss Axiovert microscope equipped with differential interference contrast (DIC) optics. Changes in capillary diameter were measured using an Openlab acquisition system and analyzed statistically using ANOVA. In addition to light microscopy the intracellular pH was analyzed in the whole mounts by ratio imaging of the pH using the special dye BCECF-AM (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluoresceinacetoxymethyl ester) and the extracellular pH using BCECF (2,7 -bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein). Pericytes of most segments of retinal capillaries are immunoreactive for alpha-smooth muscle actin (SMA). The SMA immunostaining is strong around the nucleus; the endothelial tube is visible by virtue of the slight immunoreactivity of the surrounding pericyte processes. Acetazolamide and dorzolamide showed statistically significant vasoactive effects in retinal capillaries. Vasodilation increased by up to 105% of that in control capillaries after 5, 10 and 15 min. CA inhibitors were found to be able to induce intracellular alkalization in retinal cells. After addition of dorzolamide or acetazolamide the extracellular pH decreased from 7.4 to 7.2 concomitant with diameter changes. The tube-like pattern of SMA immunoreactivity demonstrates the presence of contractile elements within the pericyte processes of the rat retina. Thus, pericytes may act as a regulation element within the retinal microcirculation. Our results further suggest that CA inhibitors are able to decrease pH in the extracellular space; however, the pH within the cells increases. The increase in capillary diameter is concomitant with these pH changes. Thus, we may conclude that CA inhibitors can relax pericytes and might improve the retinal blood supply.

Cloning and functional characterization of a new subtype of the amino acid transport system N.

We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50-56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na(+)-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li(+) tolerant, and pH sensitive. The transport mechanism involves the influx of Na(+) and amino acids coupled to the efflux of H(+), resulting in intracellular alkalization. Proline, alpha-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.

Bufo marinus bladder H-K-ATPase carries out electroneutral ion transport.

Bufo marinus bladder H-K-ATPase belongs to the Na-K-ATPase and H-K-ATPase subfamily of oligomeric P-type ATPases and is closely related to rat and human nongastric H-K-ATPases. It has been demonstrated that this ATPase transports K(+) into the cell in exchange for protons and sodium ions, but the stoichiometry of this cation exchange is not yet known. We studied the electrogenic properties of B. marinus bladder H-K-ATPase expressed in Xenopus laevis oocytes. In a HEPES-buffered solution, K(+) activation of the H-K-ATPase induced a slow-onset inward current that reached an amplitude of approximately 20 nA after 1-2 min. When measurements were performed in a solution containing 25 mM HCO at a PCO(2) of 40 Torr, the negative current activated by K(+) was reduced. In noninjected oocytes, intracellular alkalization activated an inward current similar to that due to B. marinus H-K-ATPase. We conclude that the transport activity of the nongastric B. marinus H-K-ATPase is not intrinsically electrogenic but that the inward current observed in oocytes expressing this ion pump is secondary to intracellular alkalization induced by proton transport.