Cytosolic acidification and intracellular zinc release in hippocampal neurons

In neurons exposed to glutamate, Ca2? influx triggers intracellular Zn2? release via an as yet unclear mechanism. As glutamate induces a Ca2?-dependent cytosolic acidification, the present work tested the relationships among intracellular Ca2? concentration ([Ca2?](i)), intracellular pH (pH(i) ), and [Zn2?](i). Cultured hippocampal neurons were exposed to glutamate and glycine (Glu/Gly), while [Zn2?](i), [Ca2?](i) and pH(i) were monitored using FluoZin-3, Fura2-FF, and 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Glu/Gly applications decreased pH(i) to 6.1 and induced intracellular Zn2? release in a Ca2?-dependent manner, as expected. The pH(i) drop reduced the affinity of FluoZin-3 and Fura-2-FF for Zn2?. The rate of Glu/Gly-induced [Zn2?](i) increase was not correlated with the rate of [Ca2?](i) increase. Instead, the extent of [Zn2?](i) elevations corresponded well to the rate of pH(i) drop. Namely, [Zn2?](i) increased more in more highly acidified neurons. Inhibiting the mechanisms responsible for the Ca2?-dependent pH(i) drop (plasmalemmal Ca2? pump and mitochondria) counteracted the Glu/Gly-induced intracellular Zn2? release. Alkaline pH (8.5) suppressed Glu/Gly-induced intracellular Zn2? release whereas acidic pH (6.0) enhanced it. A pH(i) drop to 6.0 (without any Ca2? influx or glutamate receptor activation) led to intracellular Zn2? release; the released Zn2? (free Zn2? plus Zn2?) bound to Fura-2FF and FluoZin-3) reached 1 μM.     

Regulation of cell-cell communication in rat bone cells: the effect of phorbol esters.

The skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inhibitor of gap junctional intercellular communication. In the present study, the inhibition of cell-cell communication by TPA has been investigated in primary bone cells from newborn rat calvaria, with an emphasis on the involvement of intracellular pH (pH(i)) and cytosolic calcium ([Ca(+2)](i)) in this process. The results show that TPA (5 x 10(-)(8) M) caused a complete inhibition of intercellular communication within 40-60 min. The intercellular communication was fully restored after overnight incubation in the presence of TPA. This effect was found to be associated with an elevation of pH(i). However, neither an increase of pH(i) alone nor exposure to TPA, under conditions preventing pH(i)-shift, were found to affect intercellular communication. It is suggested that the inhibition of intercellular communication, in the presence of TPA, depends on the pH(i)-shift itself rather than on the absolute value of pH(i). In addition, elevation of cytosolic calcium by ionomycin led to the termination of intercellular communication after 30 min. This inhibitory effect was abolished when the cells were incubated for overnight with TPA and then intracellular calcium was elevated by the addition of ionomycin. These results indicate that shift of pH(i) and the increase of intracellular calcium are involved in repression of intercellular communication by TPA.     

Generation of intracellular pH gradients in single cardiac myocytes with a microperfusion system

This study describes the use of a microperfusion system to create rapid, large regional changes in intracellular pH (pH(i)) within single ventricular myocytes. The spatial distribution of pH(i) in single myocytes was measured with seminaphthorhodafluor-1 fluorescence using confocal imaging. Changes in pH(i) were induced by local external application of NH(4)Cl, CO(2), or sodium propionate. Local application was achieved by simultaneously directing two parallel square microstreams, each 275 microm wide, over a single myocyte oriented perpendicular to the direction of flow. One stream contained the control solution, and the other contained a weak acid or base. End-to-end, stable pH(i) gradients as large as 1 pH unit were readily created with this technique. This result indicates that pH within a single cardiac cell may not always be spatially uniform, particularly when weak acid or base gradients are present, which can occur, for example, in regional myocardial ischemia. The microperfusion method should be useful for studying the effects of localized acidosis on myocyte function, estimating intracellular ion diffusion rates, and, possibly, inducing regional changes in other important intracellular ions.     

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate--the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.     

Neuronal intracellular pH directly mediates nitric oxide-induced programmed cell death.

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH(i)) regulation during nitric oxide (NO)-induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 microM), 3-morpholinosydnonimine (300 microM), or 6-(2-hyrdroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex anamine (300 microM). The pH(i) was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH(i) 7.36 +/- 0.01 to approximately 7.00 (p <.0001). Within 45 min, a biphasic alkaline response was evident, with pH(i) reaching 7.40 +/- 0.02, that was persistent for a 6-h period. To mimic the effect of NO-induced acidification, neurons were acid-loaded with ammonium ions to yield a pH(i) of 7.09 +/- 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 +/- 2% (24 h) and DNA fragmentation increased to 58 +/- 8% (24 h) (p <.0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH(i) of 7.60 +/- 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO-induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration.     

Water and electrolyte content of the myofilament phase in the chemically skinned barnacle fiber

Muscle fibers from the giant barnacle, Balanus nubilus, were placed inside the lumen of a porous glass capillary and equilibrated for 48 h in an electrolyte solution containing 2% Tween. The glass capillary prevented the chemically "skinned" fiber from swelling with a water content beyond 80%. Isotope exchange studies using 22Na, 42K, and 36Cl indicated the existence of an intermediate rate constant and compartment which varied with pH. This intermediate rate was attributed to counter-ions and co-ions in the myofilament phase. Analysis of the electrolyte composition of the fiber at pH 8 predicts that the myofilaments contain about 0.3 of the fiber water, and that a -15 mV Donnan potential exists at the myofilament surface. An open-tipped (1- micrometer) microelectrode in the skinned fiber measured a potential (similar in magnitude to the Donnan potential), which decreased and reversed sign as the pH was lowered. The measured cation contents of the fiber between pH 5 and 8 were found to be similar to the cation contents predicted from the measured Donnan potentials. The net negative charge of the myofilaments at pH 7.5 and at ionic strength 0.56 is estimated to be 41 eq per 10(5) g of dry weight.     

Endothelin-1 and PKC induce positive inotropy without affecting pHi in ventricular myocytes

It has been proposed that intracellular alkalinization underlies the enhanced contractility of ventricular myocytes exposed to endothelin (ET)-1. The effects of ET-1 on the contractility and intracellular pH (pH(i)) were examined here in cultured adult rat ventricular myocytes by employing the pH-sensitive fluorescent dye SNARF-1. Variable pH(i) changes were observed on ET-1 stimulation. Most myocytes (n = 20 of 32) did not alkalinize, but showed an approximate 60% increase in twitch amplitude in response to ET-1. In the remaining myocytes (12 of 32), ET-1 induced an increase in pH(i) by 0.05 +/- 0.02 pH units with a similar approximate 60% increase in twitch amplitude. Therefore, there was no strong correlation between ET-1-mediated positive inotropy (enhanced contractility) and intracellular alkalinization. To determine whether ET-1 contractile and pH(i) responses were mediated by protein kinase C (PKC), yellow fluorescent protein (YFP)-fused dominant negative (dn) PKC constructs were used as isoform specific inhibitors. In dn-PKC-epsilon-YFP-expressing myocytes, the ET-1-mediated positive inotropic response was greatly diminished to 13 +/- 15%, but alkalinization was still observed. Expression of dn-PKC-delta-YFP also did not block alkalinization, but in this case the positive inotropic response was still observed. In a previous study, we showed that expression of PKC-delta and PKC-epsilon caused a strong positive inotropy on stimulation with phorbol 12,13-dibutyrate (PDBu). Using this system, PDBu failed to affect pH(i) in the majority of PKC expressing myocytes despite increases in twitch amplitudes of >60%. Overall, the poor correlation of positive inotropic responses and alkalinization was observed for ET-1 with and without dn-PKC constructs and for PDBu with and without wild-type PKC constructs. These results suggest that ET-1 produces positive inotropy via PKC-epsilon by mechanisms other than intracellular alkalinization.     

Modulation of the Kir7.1 potassium channel by extracellular and intracellular pH

Inwardly rectifying K(+) (K(ir)) channels in the apical membrane of the retinal pigment epithelium (RPE) contribute to extracellular K(+) homeostasis in the distal retina by mediating K(+) secretion. Multiple lines of evidence suggest that these channels are composed of Kir7.1. Previously, we showed that native K(ir) channels in bovine RPE are modulated by changes in intracellular pH in the physiological range. In the present study, we used the Xenopus laevis oocyte expression system to investigate the pH dependence of cloned human Kir7.1 channels and several point mutants involving histidine residues in the NH(2) and COOH termini. Kir7.1 channels were inhibited by strong extracellular acidification and modulated by intracellular pH in a biphasic manner, with maximal activity at about intracellular pH (pH(i)) 7.0 and inhibition by acidification or alkalinization. Replacement of histidine 26 (H26) in the NH(2) terminus with alanine eliminated the requirement of protons for channel activity and increased sensitivity to proton-induced inhibition, resulting in maximal channel activity at alkaline pH(i) and smaller whole cell currents at resting pH(i) compared with wild-type Kir7.1. When H26 was replaced with arginine, the pH(i) sensitivity profile was similar to that of the H26A mutant but with the pK(a) shifted to a more acidic value, giving rise to whole cell current amplitude at resting pH(i) that was comparable to that of wild-type Kir7.1. These results indicate that Kir7.1 channels are modulated by intracellular protons by diverse mechanisms and suggest that H26 is important for channel activation at physiological pH(i) and that it influences an unidentified proton-induced inhibitory mechanism.     

pH-Dependence of extrinsic and intrinsic H(+)-ion mobility in the rat ventricular myocyte, investigated using flash photolysis of a caged-H(+) compound

Passive H(+)-ion mobility within eukaryotic cells is low, due to H(+)-ion binding to cytoplasmic buffers. A localized intracellular acidosis can therefore persist for seconds or even minutes. Because H(+)-ions modulate so many biological processes, spatial intracellular pH (pH(i))-regulation becomes important for coordinating cellular activity. We have investigated spatial pH(i)-regulation in single and paired ventricular myocytes from rat heart by inducing a localized intracellular acid-load, while confocally imaging pH(i) using the pH-fluorophore, carboxy-SNARF-1. We present a novel method for localizing the acid-load. This involves the intracellular photolytic uncaging of H(+)-ions from a membrane-permeant acid-donor, 2-nitrobenzaldehyde. The subsequent spatial pH(i)-changes are consistent with intracellular H(+)-mobility and cell-to-cell H(+)-permeability constants measured using more conventional acid-loading techniques. We use the method to investigate the effect of reducing pH(i) on intrinsic (non-CO(2)/HCO(3)(-) buffer-dependent) and extrinsic (CO(2)/HCO(3)(-) buffer-dependent) components of H(i)(+)-mobility. We find that although both components mediate spatial regulation of pH within the cell, their ability to do so declines sharply at low pH(i). Thus acidosis severely slows intracellular H(+)-ion movement. This can result in spatial pH(i) nonuniformity, particularly during the stimulation of sarcolemmal Na(+)-H(+) exchange. Intracellular acidosis thus presents a window of vulnerability in the spatial coordination of cellular function.     

Intracellular pH modulates inner segment calcium homeostasis in vertebrate photoreceptors

Neuronal metabolic and electrical activity is associated with shifts in intracellular pH (pH(i)) proton activity and state-dependent changes in activation of signaling pathways in the plasma membrane, cytosol, and intracellular compartments. We investigated interactions between two intracellular messenger ions, protons and calcium (Ca2(+)), in salamander photoreceptor inner segments loaded with Ca2(+) and pH indicator dyes. Resting cytosolic pH in rods and cones in HEPES-based saline was acidified by ～0.4 pH units with respect to pH of the superfusing saline (pH = 7.6), indicating that dissociated inner segments experience continuous acid loading. Cytosolic alkalinization with ammonium chloride (NH?Cl) depolarized photoreceptors and stimulated Ca2(+) release from internal stores, yet paradoxically also evoked dose-dependent, reversible decreases in [Ca2(+)](i). Alkalinization-evoked [Ca2(+)](i) decreases were independent of voltage-operated and store-operated Ca2(+) entry, plasma membrane Ca2(+) extrusion, and Ca2(+) sequestration into internal stores. The [Ca2(+)](i)-suppressive effects of alkalinization were antagonized by the fast Ca2(+) buffer BAPTA, suggesting that pH(i) directly regulates Ca2(+) binding to internal anionic sites. In summary, this data suggest that endogenously produced protons continually modulate the membrane potential, release from Ca2(+) stores, and intracellular Ca2(+) buffering in rod and cone inner segments.     

Interactions between Na+ channels and Na+-HCO3- cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pH(i)) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pH(i) from an NH(4)Cl-induced acidification in the absence of Na(+). An apparent link between resting pH(i) and Na(+)-independent pH(i) recovery was made. We observed a unique pH(i) acidification event that was induced by extracellular Na(+) addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na(+)-induced pH(i) acidification, whereas the minority (23%) demonstrated an alkalinization of pH(i) under the same circumstances. The focus of this study was placed on the Na(+)-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na(+) uptake was responsible for the intracellular acidification. Further experiments revealed that pH(i) acidification could be abolished when Na(+) was allowed entry into the cell, but the activity of an electrogenic Na(+)-HCO(3)(-) cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na(+)-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na(+) uptake occurring via an apical Na(+) channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.     

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 μM) and capsaicin (1 μM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 μM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 μM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.     

Regulation of AE2-mediated Cl- transport by intracellular or by extracellular pH requires highly conserved amino acid residues of the AE2 NH2-terminal cytoplasmic domain

We reported recently that regulation by intracellular pH (pH(i)) of the murine Cl-/HCO(3)(-) exchanger AE2 requires amino acid residues 310-347 of the polypeptide's NH(2)-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl- efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pH(o)) with unclamped or clamped pH(i), or during variation of pH(i) at constant pH(o). Wild-type AE2-mediated 36Cl- efflux was profoundly inhibited by acid pH(o), with a value of pH(o50) = 6.87 +/- 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312-347 identified the greatest acid shift in pH(o(50)) value, approximately 0.8 pH units in the mutant (A)6 342-347, but only a modest acid-shift in the mutant (A)6 336-341. Two of the six (A)6 mutants retained normal pH(i) sensitivity of 36Cl- efflux, whereas the (A)6 mutants 318-323, 336-341, and 342-347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336-347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pH(o) and to pH(i) were found independently and in concert. The E346A mutation acid-shifted the pH(o(0) value to the same extent whether pH(i) was unclamped or held constant during variation of pH(o). Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pH(o) as well as pH(i).     

Transport of ketone bodies and lactate in the sheep ruminal epithelium by monocarboxylate transporter 1

Due to intensive intracellular metabolism of short-chain fatty acids, ruminal epithelial cells generate large amounts of D-beta-hydroxybutyric acid, acetoacetic acid, and lactic acid. These acids have to be extruded from the cytosol to avoid disturbances of intracellular pH (pH(i)). To evaluate acid extrusion, pH(i) was studied in cultured ruminal epithelial cells of sheep using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Extracellular addition of D-beta-hydroxybutyrate, acetoacetate, or lactate (20 mM) resulted in intracellular acidification. Vice versa, removing extracellular D-beta-hydroxybutyrate, acetoacetate, or lactate after preincubation with the respective monocarboxylate induced an increase of pH(i). Initial rate of pH(i) decrease as well as of pH(i) recovery was strongly inhibited by pCMBS (400 microM) and phloretin (20 microM). Both cultured cells and intact ruminal epithelium were tested for the possible presence of proton-linked monocarboxylate transporter (MCT) on both the mRNA and protein levels. With the use of RT-PCR, mRNA encoding for MCT1 isoform was demonstrated in cultured ruminal epithelial cells and the ruminal epithelium. Immunostaining with MCT1 antibodies intensively labeled cultured ruminal epithelial cells and cells located in the stratum basale of the ruminal epithelium. In conclusion, our data indicate that MCT1 is expressed in the stratum basale of the ruminal epithelium and may function as a main mechanism for removing ketone bodies and lactate together with H+ from the cytosol into the blood.     

Modulation of acid-sensing ion channel currents, acid-induced increase of intracellular Ca2+, and acidosis-mediated neuronal injury by intracellular pH

Acid-sensing ion channels (ASICs), activated by lowering extracellular pH (pH(o)), play an important role in normal synaptic transmission in brain and in the pathology of brain ischemia. Like pH(o), intracellular pH (pH(i)) changes dramatically in both physiological and pathological conditions. Although it is known that a drop in pH(o) activates the ASICs, it is not clear whether alterations of pH(i) have an effect on these channels. Here we demonstrate that the overall activities of ASICs, including channel activation, inactivation, and recovery from desensitization, are tightly regulated by pH(i). In cultured mouse cortical neurons, bath perfusion of the intracellular alkalizing agent quinine increased the amplitude of the ASIC current by approximately 50%. In contrast, intracellular acidification by withdrawal of NH(4)Cl or perfusion of propionate inhibited the current. Increasing pH buffering capacity in the pipette solution with 40 mm HEPES attenuated the effects of quinine and NH(4)Cl. The effects of intracellular alkalizing/acidifying agents were mimicked by using intracellular solutions with pH directly buffered at high/low values. Increasing pH(i) induced a shift in H(+) dose-response curve toward less acidic pH but a shift in the steady state inactivation curve toward more acidic pH. In addition, alkalizing pH(i) induced an increase in the recovery rate of ASICs from desensitization. Consistent with its effect on the ASIC current, changing pH(i) has a significant influence on the acid-induced increase of intracellular Ca(2+), membrane depolarization, and acidosis-mediated neuronal injury. Our findings suggest that changes in pH(i) may play an important role in determining the overall function of ASICs in both physiological and pathological conditions.     

PH-induced changes in calcium: functional consequences and mechanisms of action in guinea pig portal vein

The effects of changing extracellular (pH(o)) and intracellular pH (pH(i)) on force and the mechanisms involved in the guinea pig portal vein were investigated to better understand the control of tone in this vessel. When pH(o) was altered, the effects on force and calcium were the same irrespective of whether force had been produced spontaneously by high-K depolarization or by norepinephrine; alkalinization increased tone, and acidification reduced it. Because pH(o) changes also lead to changes in pH(i), we determined whether the effects on force could be explained by these induced pH(i) changes. It was found, however, that only with spontaneous activity did intracellular alkalinization increase force. In depolarized preparations, force was decreased, and, with norepinephrine, force was initially decreased and then increased. Thus the effects of pH(o) cannot be explained solely by changes in pH(i). The role of the sarcoplasmic reticulum (SR) and surface membrane Ca(2+)-ATPase on the mechanism were investigated and shown not to be involved. Therefore, it is concluded that both pH(o) and pH(i) can have powerful modulatory effects on portal vein tone, that these effects are not identical, and that they are likely to be due to effects of pH on ion channels rather than the SR or plasma membrane Ca(2+)-ATPase.     

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability

? To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. ? Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). ? Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). ? The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism.     

Intracellular pH regulation in a nonmalignant and a derived malignant human breast cell line

Tumor cells in vivo often exist in an ischemic microenvironment that would compromise the growth of normal cells. To minimize intracellular acidification under these conditions, these cells are thought to upregulate H(+) transport mechanisms and/or slow the rate at which metabolic processes generate intracellular protons. Proton extrusion has been compared under identical conditions in two closely related human breast cell lines: nonmalignant but immortalized HMT-3522/S1 and malignant HMT-3522/T4-2 cells derived from them. Only the latter were capable of tumor formation in host animals or long-term growth in a low-pH medium designed to mimic conditions in many solid tumors. However, detailed study of the dynamics of proton extrusion in the two cell lines revealed no significant differences. Thus, even though the ability to upregulate proton extrusion in a low pH environment (pH(e)) may be important for cell survival in a tumor, this ability is not acquired along with the capacity to form solid tumors and is not unique to the transformed cell. This conclusion was based on fluorescence measurements of intracellular pH (pH(i)) on cells that were plated on extracellular matrix, allowing them to remain adherent to proteins to which they had become attached 24 to 48 h earlier. Proton translocation under conditions of low pH(e) was observed by monitoring pH(i) after exposing cells to an acute acidification of the surrounding medium. Proton translocation at normal pH(e) was measured by monitoring the recovery after introduction of an intracellular proton load by treatment with ammonium chloride. Even in the presence of inhibitors of the three major mechanisms of proton translocation (sodium-proton antiport, bicarbonate transport, and proton-lactate symport) together with acidification of their medium, cells showed only about 0.4 units of reduction in pH(i). This was attributed to a slowing of metabolic proton generation because the inhibitors were shown to be effective when the same cells were given an intracellular acidification.