Intracellular pH regulation in rat round spermatids

Intracellular pH has been shown to be an important physiological parameter in cell cycle control and differentiation, aspects that are central to the spermatogenic process. However, the pH regulatory mechanisms in spermatogenic cells have not been systematically explored. In this work, measuring intracellular pH (Hi) with a fluorescent probe (BCECF), membrane potential with a fluorescent lipophilic anion (bisoxonol), and net movement of acid using a pH-stat system, we have found that rat round spermatids regulate pHi by means of a V-type H⁺-ATPase, a HCO ^{3 ?} entry pathway, a Na⁺ HCO3^?dependent transport system, and a putative proton conductive pathway. Rat spermatids do not have functional base extruder transport systems. These pH regulatory characteristics seem specially designed to withstand acid challenges, and can generate sustained alkalinization upon acid exit stimulation.     

Intracellular pH regulation in the early embryo

Intracellular pH (pHi) regulation is a homeostatic function of all cells. Additionally, the plasma membrane-based transporters controlling pHi are involved in growth factor activation, cell proliferation and salt transport – all processes active in early embryos. pHi regulation in the early embryos of many species exhibits unique features: in mouse preimplantation embryos, mechanisms for correcting excess acid apparently are inactive, while excess base is removed by the mechanism common in differentiated cells. Additionally, unlike differentiated cells, mouse preimplantation embryos are highly permeable to H⁺ until the blastocyst stage, where the epithelial cells surrounding the embryo are impermeable. In several non-mammalian species, of which the best-studied is sea urchin, cytoplasmic alkalinization at fertilization is necessary for development of the embryo, and elevated pHi must be maintained during early development. Thus, pHi regulatory mechanisms appear to be important for early embryo development in many species.     

Intracellular pH regulation in hepatocytes isolated from three teleost species.

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Intracellular pH regulation during spreading of human neutrophils

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.     

Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Intramolecular interactions mediate pH regulation of connexin43 channels.

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.     

Intracellular pH regulation in maize root tips exposed to ammonium at high external pH

Ammonium-induced changes in the cytoplasmic and vacuolar pH values of excised maize (Zea mays L.) root tips, measured by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy, were correlated with the ammonium content of the tissue, determined by 14N NMR. Calculations based on these measurements indicated that the pH changes observed during exposure to 10 mM ammonium for 1 h at pH 9.0, and in the recovery following the removal of the external ammonium supply, were largely determined by the influx and efflux of the weak base NH3. Carboxylate synthesis, detected by both in vivo 13C NMR and the incorporation of [14C]bicarbonate, was stimulated by the ammonium-induced alkalinization of the root tips, but the contribution that this proton-generating process made to pH regulation during and after the ammonium treatment was quantitatively insignificant. Similarly, ammonium assimilation, which was shown to occur via the proton-generating glutamine synthetase/glutamate synthase pathway using in vivo 15N NMR, was also quantitatively insignificant in comparison with the large changes in ammonium content that occurred during the ammonium treatment and subsequent recovery. The results are discussed in relation to several recent studies in which ammonium was used to perturb intracellular pH values, and it is argued (i) that a new method for probing the subcellular compartmentation of amino acids, based on an ammonium-induced alkalinization of the cytoplasm may be difficult to implement in dense heterogeneous tissues; and (ii) that observations on the apparently proton-consuming effect of ammonium assimilation in rice root hairs may actually reflect unusually rapid assimilation.     

Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange

Using pH-sensitive microelectrodes to measure intracellular pH (pHi) in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum, we have found that when cells are acid-loaded by pretreatment with NH+4 in a nominally HCO3--free Ringer, pHi spontaneously recovers with an exponential time course. This pHi recovery, which is indicative of active (i.e., uphill) transport, is blocked by removal of Na+ from both the luminal and basolateral (i.e., bath) solutions. Re-addition of Na+ to either the lumen or the bath results in a full pHi recovery, but at a lower-than-normal rate; the maximal rate is achieved only with Na+ in both solutions. The diuretic amiloride reversibly inhibits the pHi recovery when present on either the luminal or basolateral sides, and has its maximal effect when present in both solutions. The pHi recovery is insensitive to stilbene derivatives and to Cl- removal. A transient rise of intracellular Na+ activity accompanies the pHi recovery; there is no change of intracellular Cl- activity. These data suggest that these proximal tubule cells have Na-H exchangers in both the luminal and basolateral membranes.     

Intracellular pH and the metabolic status of dormant and developing Artemia embryos

Encysted embryos of the brine shrimp Artemia salina have long been known to enter profound dormancy under anaerobic conditions. Utilizing in vivo³¹P nuclear magnetic resonance, we show that the reversible transitions between anaerobic dormancy and aerobic development are accompanied by large (>1 unit) intracellular pH changes, the more acid pH being associated with the dormant state. Furthermore, dormant cyst intracellular pH is independent of that of the buffer, suggesting the potential for pH-mediated regulation of enzyme activities during dormancy. An example concerning cyst nucleotide metabolism is discussed.     

Intracellular pH regulation in neurons from chemosensitive and nonchemosensitive regions of Helix aspersa

We used 2',7'-bis(carboxyethyl)-5(6)-carboxyflourescein (BCECF), a pH-sensitive fluorescent dye, to study intracellular pH (pH(i)) regulation in neurons in CO(2) chemoreceptor and nonchemoreceptor regions in the pulmonate, terrestrial snail, Helix aspersa. We studied pH(i) during hypercapnic acidosis, after ammonia prepulse, and during isohydric hypercapnia. In all treatment conditions, pH(i) fell to similar levels in chemoreceptor and nonchemoreceptor regions. However, pH(i) recovery was consistently slower in chemoreceptor regions compared with nonchemoreceptor regions, and pH(i) recovery was slower in all regions when extracellular pH (pH(e)) was also reduced. We also studied the effect of amiloride and DIDS on pH(i) regulation during isohydric hypercapnia. An amiloride-sensitive mechanism was the dominant pH(i) regulatory process during acidosis. We conclude that pH(e) modulates and slows pH(i) regulation in chemoreceptor regions to a greater extent than in nonchemoreceptor regions by inhibiting an amiloride-sensitive Na(+)/H(+) exchanger. Although the phylogenetic distance between vertebrates and invertebrates is large, similar results have been reported in CO(2)-sensitive regions within the rat brain stem.     

Relationship of hepatic cholate transport to regulation of intracellular pH and potassium.

Modulation of hepatic cholate transport by transmembrane pH-gradients and during interferences with the homeostatic regulation of intracellular pH and K+ was studied in the isolated perfused rat liver. Within the concentration range studied uptake into the liver was saturable and appeared to be associated with release of OH- and uptake of K+. Perfusate acidification ineffectually stimulated uptake. Application of NH4Cl caused intracellular alkalinization, release of K+ and stimulation of cholate uptake, withdrawal of NH4Cl resulted in intracellular acidification, regain of K+ and inhibition of cholate uptake. Inhibition of Na+/H(+)-exchange with amiloride reduced basal release of acid equivalents into the perfusate, initiated K(+)-release, and inhibited both, control cholate uptake and its recovery following intracellular acidification. K(+)-free perfusion caused K(+)-release and inhibited cholate uptake. K(+)-readmission resulted in brisk K(+)-uptake and recovery of cholate transport. Both effects were inhibited by amiloride. Interference with cholate transport through modulation of pH homeostasis by diisothiocyanostilbenedisulfonate (DIDS) could not be demonstrated because DIDS affected bile acid transport directly. Biliary bile acid secretion was stimulated by intracellular alkalinization and by activation of K(+)-transport. Uncoupling of the mutual interference between pH-dependent cholate uptake and K(+)-transport by amiloride indicates tertiary active transport of cholate. In this, Na+/K(+)-ATPase provides the transmembrane Na(+)-gradient to sustain Na+/H(+)-exchange which maintains the transmembrane pH-gradient and thus supports cholate uptake. Effects of canalicular bile acid secretion are consistent with a saturable, electrogenic transport.     

Nutritional and metabolic regulation of brown and beige adipose tissues

Journal of Physiology and Biochemistry -     

Intracellular pH of Thermoplasma acidophila.

Thermoplasma acidophila, a mycoplasma-like organism, was grown at 56 degrees C and pH 2. The intracellular pH of this organism is close to neutral as measured by the distribution of a radioactive weak organic acid, 5,5-dimethyl-2,4-oxazolidinedione, across the plasma membrane. The cell can maintain the pH gradient when subjected to heat or to metabolic inhibitors. Our experiments seem to indicate that a major portion of the pH gradient is not maintained by active processes, but rather by a Donnan potential across the cell membrane.     

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.