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 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 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 ventral horn neurones cultured from embryonic rat spinal cord

Summary

We have isolated and characterized a cDNA from the marine sponge Geodia cydonlum coding for a new member of the tyrosine protein kinase (TK) family. The cDNA encodes a protein of M_r = 68 710, termed GCTK, which is homologous to class II receptor tyrosine kinases (RTKs). GCTK contains conserved amino acids (aa) characteristic of all protein kinases, and the sequences DLATRN and PIRWMATE which are highly specific for TKs. Furthermore, the sequence N-L-Y-x(3)-Y-Y-R Is highly homologous to the sequence D-[LIV]-Y-x(3)-Y-Y-R found only in class II RTKs. The sponge TK, when compared with mammalian class II RTKs, shows maximum 31% homology in the TK domain indicating that this the oldest member of class II RTK started to diverge from the common ancestral protein kinase 650 million years ago. Using GCTK as a probe we identified three mRNA signals ranging from 2μ6 to 0μ6 kb. Kinase activity was localized only in the cell membranes from G. cydonium (M_r = 65 000), and was not detected in the cytosol of this organism. Antibodies raised against a synthetic peptide, corresponding to the aa residues within the catalytic domain of the sponge TK, recognized strongly two proteins of M_r = 65 000; these proteins, present in membrane fractions, also bound to the anti-phosphotyrosine antibody. These data suggest that the TK cloned from the sponge is a membrane-associated 65 kDa protein. Moreover these results demonstrate that RTKs are present from the lowest group of multicellular eukaryotes, sponges, to mammals, and may suggest that RTKs are involved in a signal transduction pathway.     

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.     

Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3- transport

We have used pH-, Na-, and Cl-sensitive microelectrodes to study basolateral HCO3- transport in isolated, perfused proximal tubules of the tiger salamander Ambystoma tigrinum. In one series of experiments, we lowered basolateral pH (pHb) from 7.5 to 6.8 by reducing [HCO3-]b from 10 to 2 mM at a constant pCO2. This reduction of pHb and [HCO3-]b causes a large (approximately 0.35), rapid fall in pHi as well as a transient depolarization of the basolateral membrane. Returning pHb and [HCO3-]b to normal has the opposite effects. Similar reductions of luminal pH (pHl) and [HCO3-]l have only minor effects. The reduction of [HCO3-]b and pHb also produces a reversible fall in aiNa. In a second series of experiments, we reduced [Na+]b at constant [HCO3-]b and pHb, and also observed a rapid fall in pHi and a transient basolateral depolarization. These changes are reversed by returning [Na+]b to normal. The effects of altering [Na+]l in the presence of HCO3-, or of altering [Na+]b in the nominal absence of HCO3-, are substantially less. Although the effects on pHi and basolateral membrane potential of altering either [HCO3-]b or [Na+]b are largely blocked by 4-acetamido-4- isothiocyanostilbene-2,2'-disulfonate (SITS), they are not affected by removal of Cl-, nor are there accompanying changes in aiCl consistent with a tight linkage between Cl- fluxes and those of Na+ and HCO3-. The aforementioned changes are apparently mediated by a single transport system, not involving Cl-. We conclude that HCO3- transport is restricted to the basolateral membrane, and that HCO3- fluxes are linked to those of Na+. The data are compatible with an electrogenic Na/HCO3 transporter that carries Na+, HCO3-, and net negative charge in the same direction.     

Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.     

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 and metabolic interactions in hepatic tissues.

Intracellular pH (pHi) regulation in the vertebrate liver relies heavily on ionic transport mechanisms. Liver, in common with many tissues, has plasma membrane Na(+)-H+ and Cl(-)-HCO3- electroneutral exchangers which work in opposition to tightly control pHi. Mammalian livers also possess electrogenic Na(+)-HCO3- exchangers, capable of base uptake, which, when coupled to pHi-mediated changes in membrane potential, probably confer an additional measure of pHi control, compared to fish livers, where the transporter appears to be functionally absent. It is suggested that this may be a fundamental difference between aquatic and aerial breathing. pHi regulation has barely been examined in invertebrate hepatic tissues, but already some interesting differences are apparent. Notably, an electrogenic 2Na(+)-1H+ acid-extrusion system is present in apical membranes of crustacean hepatopancreas. Despite these ionic control systems, complex acid-base disturbances (e.g., "metabolic" acidosis) have been known for some time to influence hepatic metabolism in vertebrates, but few studies have carefully examined the independent effects of the acid-base variables involved. Thus mechanistic explanations for the effects of acid-base disturbances are scarce. Ureogenesis in mammals has been well studied, and several pH-related mechanisms are evident. In contrast, the pH-insensitivity of ureogenesis in fish liver may represent a second difference between aquatic and terrestrial species. In summary, by virtue of its metabolic diversity, liver represents a potentially important organ in acid-base balance, and an interesting study tissue for interrelationships between metabolism and acid-base balance.     

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.     

An Na(+)-independent short-chain fatty acid transporter contributes to intracellular pH regulation in murine colonocytes

Short-chain fatty acids (SCFAs) are the major anions in the colonic lumen. Experiments studied how intracellular pH (pHi) of isolated colonocytes was affected by exposure to SCFAs normally found in the colon. Isolated crypt fragments were loaded with SNARF-1 (a fluorescent dye with pH-sensitive excitation and emission spectra) and studied in a digital imaging microscope. Intracellular pH was measured in individual colonocytes as the ratio of fluorescence intensity in response to alternating excitation wavelengths (575/505 nm). After exposure to 65 mM acetate, propionate, n-butyrate, or iso-butyrate in isosmotic Na(+)- free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pHi alkalinization (Na(+)-independent pHi recovery). Upon subsequent removal of the SCFA, pHi alkalinized beyond the starting pHi (a pHi overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and pHi overshoot are explained by transmembrane influx and efflux of nonionized SCFA, respectively. The basis for the pHi overshoot is shown to be accumulation of propionate during pHi alkalinization. The Na(+)-independent pHi recovery (a) demonstrates saturable propionate activation kinetics; (b) demonstrates substrate specificity for unmodified aliphatic carbon chains; (c) occurs after exposure to SCFAs of widely different metabolic activity, (d) is electroneutral; and (e) is not inhibited by changes in the K+ gradient, Cl- gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), alpha-cyano-4-hydroxycinnamate (4 mM), or probenicid (1 mM). Results suggest that most mouse colonocytes have a previously unreported SCFA transporter which mediates Na(+)-independent pHi recovery.     

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 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.