Intracellular pH of bovine sperm increases during capacitation

The effect of heparin-induced capacitation on the intracellular pH (pH_i) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2′,7′-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). The pH_i of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pH_i did not differ between the sperm head and mid-piece (P > 0.05). An increase in sperm head pH_i was seen in heparin-treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P < 0.05). At 5 h of incubation, the pH_i in heparin-treated sperm was 6.92 ± 0.07, while control-treated sperm pH_i was 6.70 ± 0.03. Initially a normal frequency distribution was seen for sperm pH_i in both heparin- and control-treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pH_i in both samples but was more dispersed for the heparin-treated sperm. Following an NH₄Cl-induced alkaline load, the pH_i of both control- and heparin-treated sperm recovered toward the resting pH_i with a half-time of recovery of 1.5–1.7 min. The recovery of sperm pH_i was not due to leakage of NH⁴⁺ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pH_i recovery (v_i) was dependent on pH_i and decreased as pH_i decreased. Capacitation by heparin was associated with an 81% decrease in v_i at a pH_i of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 ± 8 mM/pH unit. Results demonstrate that both the regulation of pH_i and resting pH_i were altered during capacitation of bovine sperm by heparin. © 1995 Wiley-Liss, Inc.     

Effects of NH<Subscript>4</Subscript>CL application and removal on astrocytes and endothelial cells

Background

Hepatic encephalopathy (HE) is a complex disorder associated with increased ammonia levels in the brain. Although astrocytes are believed to be the principal cells affected in hyperammonemia (HA), endothelial cells (ECs) may also play an important role by contributing to the vasogenic effect of HA.

Methods

Following acute application and removal of NH₄Cl on astrocytes and endothelial cells, we analyzed pH changes, using fluorescence imaging with BCECF/AM, and changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), employing fluorescence imaging with Fura-2/AM. Using confocal microscopy, changes in cell volume were observed accompanied by changes of [Ca²⁺]_i in astrocytes and ECs.

Results

Exposure of astrocytes and ECs to 1 – 20 mM NH₄Cl resulted in rapid concentration-dependent alkalinization of cytoplasm followed by slow recovery. Removal of the NH₄Cl led to rapid concentration-dependent acidification, again followed by slow recovery. Following the application of NH₄Cl, a transient, concentration-dependent rise in [Ca²⁺]_i in astrocytes was observed. This was due to the release of Ca²⁺ from intracellular stores, since the response was abolished by emptying intracellular stores with thapsigargin and ATP, and was still present in the Ca²⁺-free bathing solution. The removal of NH₄Cl also led to a transient concentration-dependent rise in [Ca²⁺]_i that resulted from Ca²⁺ release from cytoplasmic proteins, since removing Ca²⁺ from the bathing solution and emptying intracellular Ca²⁺ stores did not eliminate the rise. Similar results were obtained from experiments on ECs. Following acute application and removal of NH₄Cl no significant changes in astrocyte volume were detected; however, an increase of EC volume was observed after the administration of NH₄Cl, and EC shrinkage was demonstrated after the acute removal of NH₄Cl.

Conclusions

This study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE.

     

Properties of a Novel pH-dependent Ca2+ Permeation Pathway Present in Male Germ Cells with Possible Roles in Spermatogenesis and Mature Sperm Function

Rises of intracellular Ca²⁺ ([Ca²⁺]_i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca²⁺]_i and intracellular pH (pH_i) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca²⁺ channels modulated by pH_i in the origin of these Ca²⁺ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca²⁺]_i on identified spermatogenic cells from mouse adult testes. In BCECF [(2′,7′)-bis(carboxymethyl)- (5,6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30–60 s) application of 25 mM NH₄Cl increased pH_i by ∼1.3 U from a resting pH_i ∼6.65. A steady pH_i plateau was maintained during NH₄Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca²⁺]_i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca²⁺ influx or intracellular Ca²⁺ release demonstrated that the majority of the Ca²⁺ rise results from plasma membrane Ca²⁺ influx, although a small component likely to result from intracellular Ca²⁺ release was occasionally observed. Ca²⁺ transients potentiated with repeated NH₄Cl applications, gradually obliterating the initial [Ca²⁺]_i drop. The pH-sensitive Ca²⁺ permeation pathway allows the passage of other divalents (Sr²⁺, Ba²⁺, and Mn²⁺) and is blocked by inorganic Ca²⁺ channel blockers (Ni²⁺ and Cd²⁺), but not by the organic blocker nifedipine. The magnitude of these Ca²⁺ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca²⁺ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca²⁺ channel also present in spermatogenic cells. We postulate that the Ca²⁺ permeation pathway regulated by pH_i, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca²⁺ influx required for initiating the acrosome reaction and perhaps other important sperm functions.     

Growth inhibition of hybridoma cells by ammonium ion: correlation with effects on intracellular pH

The effect of NH₄Cl addition on intracellular pH (pH _i ) was determined by flow cytometric measurements of the fluorescence of a pH-sensitive dye. The effects of NH₄Cl on growth were determined for batch growth of cells in flasks in an incubator. The addition of NH₄Cl caused a cytoplasmic acidification. A new lower steady-state value of pH _i was attained within 20–40 min of NH₄Cl addition. A correlation was found between the effects of NH₄Cl on growth and on pH _i : whereas 3 mM NH₄Cl had little effect on growth and on pH _i , 10 mM NH₄Cl caused a substantial growth inhibition and a pH _i decrease of 0.2–0.3 units. The effects of NH₄Cl on growth and on pH _i were found to be independent of the external pH value (pH _e over the range 6.8 to 7.6, except that 10 mM NH₄Cl was more toxic at pH _e 7.6. The addition of NH₄Cl caused an increase in the average cell volume at pH _e 7.6, but had no effect on the average cell volume at pH _e 's 6.8 and 7.2. For comparison, the effects of pH _e alone on growth and on pH _i were determined. There was little difference in cell growth at pH _e 's 6.8, 7.2 and 7.6. At pH _e 6.6, there was a substantial growth inhibition. Some measurements of the effects of pH _e on pH _i were made, although the steady-state value of pH _i as a function of pH _e was not determined due to limitations in the pH _i -measuring technique. These measurements showed that pH _i remained constant from pH _e 7.6 to 6.8, but fell by 0.2 units at pH _e 6.6, in agreement with the growth results.     

NH4 + and NO3 − requirement for wheat somatic embryogenesis

The influence of three nitrogen salts: NH₄NO₃, KNO₃ and NH₄Cl on wheat in vitro cultures was investigated. Both NO ₃ ⁻ and NH ₄ ⁺ ions were indispensable for proliferation of embryogenic calli and development of wheat somatic embryos. It is possible to obtain wheat somatic embryos when the medium is enriched with NH₄NO₃ only as a source of inorganic nitrogen. The results of the statistical analysis showed that the level of NH₄NO₃ and KNO₃ in the medium had a great influence on the efficiency of somatic embryogenesis. We observed tendency that calli on media containing 50 mM NH₄NO₃ and 0 to 20 mM KNO₃ turned out to be more embryogenic than on control MS medium. High concentrations of KNO₃- 100 mM inhibited somatic embryogenesis, while 100 mM NH₄NO₃ did not. The level of total N did not have significant influence on wheat somatic embryogenesis. Ratio NO ₃ ⁻ :NH ₄ ⁺ also turned out to be not substantial. We observed that mutual connection of concentration levels between NH₄NO₃ and KNO₃ and between NH₄Cl and KNO₃ was more important. The efficiency of somatic embriogenesis obtained in the experiment with NH₄Cl and KNO₃ was significantly lower than in experiment with NH₄NO₃ and KNO₃.     

Characterization of DNA uptake by the cyanobacterium Anacystis nidulans

Summary The binding and uptake of nick-translated ³²P-labeled pBR322 by Anacystis nidulans 6301 have been characterized. Both processes were considerably enhanced in permeaplasts compared to cells. The breakdown of labeled DNA was not correlated with binding or uptake by permeaplasts or cells. Uptake of DNA by permeaplasts was unaffected by: Mg²⁺ or Ca²⁺, light, or inhibitors of photophosphorylation such as valinomycin or gramicidin D in the presence or absence of NH₄Cl. ATP at 2.5–10 mM inhibited both binding and uptake of labeled DNA by permeaplasts of A. nidulans whereas the ATP analog adenyl-5-yl imido-diphosphate was non-inhibitory in the same concentration range. In contrast to transformation of A. nidulans 6301 cells to ampicillin-resistance by pBR322, transformation to kanamycin-resistance by the plasmid pHUB4 was considerably enhanced in the dark. The transformation efficiency for permeaplasts by the plasmid pCH1 was 59% and 8% in the dark and light, respectively, whereas transformation of permeaplasts by pBR322 at an efficiency of 16% was absolutely light-dependent.     

Calcium Dynamics During Physiological Acidification in Xenopus Oocyte

Interplays between intracellular pH (pHi) and calcium ([Ca²⁺]_i) variations remain unclear, though both proton and calcium homeostasis changes accompany physiological events such as Xenopus laevis oocyte maturation. In this report, we used NH₄Cl and changes of extracellular pH (pHe) to acidify the cytosol in a physiological range. In oocytes voltage-clamped at −80 mV, NH₄Cl triggered an inward current, the main component of which is a Ca²⁺-dependent chloride current. Calcium imaging confirmed that NH₄Cl provoked a [Ca²⁺]_i increase. The mobilized sources of calcium were discriminated using the triple-step protocol as a means to follow both the calcium-activated chloride currents (ICl-Ca) and the hyperpolarization- and acid-activated nonselective cation current (I_In). These currents were stimulated during external addition of NH₄Cl. This upregulation was abolished by BAPTA-AM, caffeine and heparin. By both buffering pHi changes with MOPS and by inhibiting calcium influx with lanthanum, intracellular acidification, initiated by NH₄Cl and extracellular acidic medium, was shown to trigger a [Ca²⁺]_i increase through both calcium release and calcium influx. The calcium pathways triggered by pHe changes are similar to those activated by NH₄Cl, thus suggesting that there is a robust signaling mechanism allowing the cell to adjust to variable environmental conditions.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

Lack of effect of NH4Cl on protein synthesis in cultured fibroblasts

In experiments with isolated hepatocytes, Seglen [1] has shown that in the combined presence of NH₄Cl and high concentrations of valine, incorporation of this amino acid into cell protein is inhibited. He has proposed that NH₄Cl, in addition to inhibiting protein degradation in lysosomes, inhibits protein synthesis in these cells as part of a general toxic effect. To determine if NH₄Cl inhibits protein synthesis in cultured cells we incubated rat embryo fibroblasts, prelabeled with [¹⁴C]leucine, in the presence of 10 mM NH₄Cl and 15 mM leucine in both growth and serum-free media. We did not detect any effect of NH₄⁺ on protein synthesis or cell growth over a 3-day period. A partial inhibition of protein degradation was observed, particularly during the first 24 h of the experiment. In pulse-labeling experiments, NH₄Cl had no effect on the incorporation of [³H]leucine in the media. High concentrations of leucine, however, reduced re-utilization of endogenously derived leucine and inhibited the transport of valine into the cellular acid-soluble pool.These experiments show that at least in cultured fibroblasts 10 mM NH₄Cl shows no significant toxicity beyond an inhibition of lysosomal function. In addition these data suggest the possibility that high chase concentrations of one amino acid in the medium may be saturating a common transport mechanism, in effect reducing the transport of other amino acids utilizing this mechanism. A combined blockade by both NH₄Cl and a high concentration of a single amino acid may in certain sensitive cells result in a significant reduction in protein synthesis.     

Growth and fermentation responses of Selenomonas ruminantium to limiting and non-limiting concentrations of ammonium chloride

 The objective of this study was to assess fermentation product, growth rate and growth yield responses of Selenomonas ruminantium HD₄ to limiting and non-limiting ammonia concentrations. The ammonia half-inhibition constant for S. ruminantium in batch culture was 296 mM. Cells were grown in continuous culture with a defined ascorbate-reduced basal medium containing either 0.5, 5, 25, 50, 100 or 200 mM NH₄Cl and dilution rates were 0.07, 0.14, 0.24 or 0.40 h^-1. Ammonia was the growth-limiting nutrient when 0.5 mM NH₄Cl was provided and the half-saturation constant was 72 μM. Specific rates of glucose utilization and fermentation acid carbon formation were highest for 0.5 mM NH₄Cl. Lactate production (moles per mole of glucose disappearing) increased at the fastest dilution rate (0.40 h^-1) for 5.0 mM NH₄Cl while acetate and propionate decreased when compared to slower dilutions (0.07 and 0.14 h^-1). Lactate production remained low while acetate and propionate remained high for all dilution rates when NH₄Cl concentrations were 25 mM or greater. Yield (Y _Glc and Y _ATP) were nearly doubled when NH₄Cl was increased from 0.5 mM (25.1 g cells/mol glucose used and 13.9 g cells/mol ATP produced respectively) to the higher concentrations. Y _Glc was highest at 25 mM and 50 mM NH₄Cl (48.2 cells/mol and 43.1 cells/mol respectively) as was Y _ATP (23.2 cells/mol and 20.8 cells/mol respectively). Y _NH3 was highest at the lowest NH₄Cl concentration. The maximal fermentation product formation rate occurred at a growth-limiting ammonia concentration, while maximal glucose and ATP bacterial yields occurred at non-growth-limiting ammonia concentrations. Given the growth response of this ruminal bacterium, it is possible that maximization of ruminal bacterial yield may necessitate sacrificing the substrate degradation rate and vice versa. Received: 5 December 1995/Received revision: 2 April 1996/Accepted: 22 April 1996     

Energy Supply and Light-enhanced Chloride Uptake in Wheat Laminae

The light-supported component of ³⁶Cl uptake from 5 mM K³⁶Cl by green laminae, either chopped or vacuum-infiltrated, of Triticum aestivum L. seedlings has been determined by subtraction of dark uptake values from light uptake values and the energy sources for the uptake elucidated on the basis of the effects of 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU), carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A₁ (AA), and N₂ on light and dark uptake. The light-supported Cl⁻¹ uptake is shown to be partially inhibited by DCMU or AA but unaffected or stimulated by FCCP or N₂. There is no additive effect on inhibition caused by DCMU + N₂ or FCCP + AA but there is an added inhibition caused by DCMU + AA, DCMU + FCCP, and by FCCP or AA in anaerobic conditions. The effect of these inhibitors on photosynthetic gas exchange of chopped tissue has also been determined. On the basis of the results it is concluded that the uptake of Cl⁻, supported in the dark by oxidative phosphorylation, is enhanced by light and may be supported by cyclic and non-cyclic electron-flow energy. Uptake is not obligatorily linked to any one energetic pathway and there may be switching from one source to another.     

Kinetic Studies of Ammonia Monooxygenase Inhibition in Nitrosomonas europaea by Hydrocarbons and Halogenated Hydrocarbons in an Optimized Whole-Cell Assay

The inhibitory effects of 15 hydrocarbons and halogenated hydrocarbons on NH₃ oxidation by ammonia monooxygenase (AMO) in intact cells of the nitrifying bacterium Nitrosomonas europaea were determined. Determination of AMO activity, measured as NO₂^- production, required coupling of hydroxylamine oxidoreductase (HAO) activity with NH₃-dependent NH₂OH production by AMO. Hydrazine, an alternate substrate for HAO, was added to the reaction mixtures as a source of reductant for AMO. Most inhibitors exhibited competitive or noncompetitive inhibition patterns. The competitive character generally decreased (K_iE/K_iES increased) as the molecular size of the inhibitors increased. For example, CH₄ and C₂H₄ were competitive inhibitors of NH₃ oxidation, whereas the remaining alkanes (up to C₄) and monohalogenated (Cl, Br, I) alkanes were noncompetitive. Oxidation of C₂H₅Br (noncompetitive) increased as the NH₄⁺ concentration increased up to 40 mM, whereas oxidations of inhibitors with competitive character (K_iE K_iES) were diminished at 40 mM NH₄⁺. Multichlorinated compounds produced nonlinear Lineweaver-Burk plots. Iodinated alkanes (CH₃I, C₂H₅I) and C₂Cl₄ were potent inhibitors of NH₃ oxidation. Maximum rates of NH₃, C₂H₄, and C₂H₆ oxidations were approximately equivalent, suggesting a common rate-determining step. These data support an active-site model for AMO consisting of an NH₃-binding site and a second site that binds noncompetitive inhibitors, with oxidation occurring at either site.     

Transfer of Sensitivity to Tumor Promoters by Transfection of DNA from Sensitive into Insensitive Mouse JB6 Epidermal Cells

Sensitivity to promotion of transformation by tumor promoters in mouse epidermal JB6 cells appears to have a genetic basis since the phenotypes of both promotable and nonpromotable JB6 cells derived from a common parent line are stable. Hybridization of promotable (P⁺) and nonpromotable (P⁻) cells previously indicated that promotability appears to behave as a dominant trait. These results suggest that it should be possible to find DNA sequences which specify sensitivity to promotion of anchorage independence by 12-o-tetradecanoyl-phorbol-13-acetate (TPA). Cellular DNA isolated from one of two P⁺ lines, JB6 Cl 41 or JB6 Cl 22, was CaPO₄ precipitated and used to transfect the P⁻ cell line JB6 Cl 30. At 7 days posttransfection, the cells were suspended in agar with or without TPA at 1.6 × 10⁻⁸ M and assayed 10 days later for TPA-dependent colony formation. Untreated or Cl 30 DNA-treated P⁻ JB6 Cl 30 cells yielded 40 to 50 colonies per 10⁵ cells. In contrast, transfection of Cl 30 cells with “P⁺ DNA” derived from either Cl 41 or Cl 22 yielded 200 to 500 TPA-induced colonies per 10⁵ cells, or a five- to eightfold enhancement of promotability. The enhanced promotability obtained after transfection with P⁺ DNA was stable, as judged by the retention of promotability for at least eight passages in cell lines derived from TPA-induced agar colonies. Other transfectants showed irreversible transformation by TPA, as observed in the parental P⁺ lines. When NIH 3T3 cells instead of the putative preneoplastic JB6 Cl 30 cells were used as recipients for transfection of P⁺ DNA, no evidence for acquisition of promotability was obtained. P⁻ JB6 Cl 25, like Cl 30, also permitted expression of transfected P⁺ DNA. These results suggest that sensitivity to phorbol ester promotion of transformation in JB6 cells is determined by DNA sequence(s) present in the P⁺ DNA and requires recipient cells of the appropriate phenotype for expression.     

Effect of form and level of applied nitrogen on nitrogenase and nitrate reductase activities in faba beans

The effects of nitrogen applied at increasing levels of 0, 4, 8, 16 and 32 mM N (KNO₃ or NH₄Cl) were studied in faba bean (Vicia faba) nodulated byRhizobium leguminosarum bv.viceae RCR lool. Nitrogenase activity was higher at 4 and 8 mM N than the zero N treatment (control), but 16 and 32 mM N significantly reduced the efficiency of nodule functions. Nitrate reductase activities (NRA) of leaves, stems, roots, nodules and nodule fractions (bacteroid and cytosol) were increased with rising the NO₃ ^? or NH₄ ⁺ levels. NRA decreased in the order of nodules>leaves>stems>roots. Cytosolic NR was markedly higher than that recorded in the bacteroid fractions. Nitrate levels were linearly correlated to NRA of nodules. Accumulation of NO₂ ^? within nodules suggests that NO₂ ^? inhibits nodule’s activity after feeding plants with NO₃ ^? or NH₄ ⁺.     

Concentration-Dependent Effects on Intracellular and Surface pH of Exposing <Emphasis Type="Italic">Xenopus</Emphasis> oocytes to Solutions Containing NH<Subscript>3</Subscript>/NH<Subscript>4</Subscript><Superscript>+</Superscript>

Others have shown that exposing oocytes to high levels of (10–20 mM) causes a paradoxical fall in intracellular pH (pH_i), whereas low levels (e.g., 0.5 mM) cause little pH_i change. Here we monitored pH_i and extracellular surface pH (pH_S) while exposing oocytes to 5 or 0.5 mM NH₃/NH₄ ⁺. We confirm that 5 mM causes a paradoxical pH_i fall (−ΔpH_i ≅ 0.2), but also observe an abrupt pH_S fall (−ΔpH_S ≅ 0.2)—indicative of NH₃ influx—followed by a slow decay. Reducing [NH₃/NH₄ ⁺] to 0.5 mM minimizes pH_i changes but maintains pH_S changes at a reduced magnitude. Expressing AmtB (bacterial Rh homologue) exaggerates −ΔpH_S at both levels. During removal of 0.5 or 5 mM NH₃/NH₄ ⁺, failure of pH_S to markedly overshoot bulk extracellular pH implies little NH₃ efflux and, thus, little free cytosolic NH₃/NH₄ ⁺. A new analysis of the effects of NH₃ vs. NH₄ ⁺ fluxes on pH_S and pH_i indicates that (a) NH₃ rather than NH₄ ⁺ fluxes dominate pH_i and pH_S changes and (b) oocytes dispose of most incoming NH₃. NMR studies of oocytes exposed to ¹⁵N-labeled show no significant formation of glutamine but substantial accumulation in what is likely an acid intracellular compartment. In conclusion, parallel measurements of pH_i and pH_S demonstrate that NH₃ flows across the plasma membrane and provide new insights into how a protein molecule in the plasma membrane—AmtB—enhances the flux of a gas across a biological membrane.

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH_i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na⁺-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH_i is more acidic than the external medium (pH_o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na⁺ to sperm suspended in Na⁺-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH_i by 0.4–0.5 pH units. That this pH_i change is the causal trigger of motility was suggested by experiments using NH₄Cl and nigericin, which increased the pH_i and resulted in activation of motility in the absence of Na⁺. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH_i was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca²⁺ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH_i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca²⁺ uptake. Either mitochondrial agents or the removal of external Ca²⁺ could also block this pH_i change suggesting a similar mechanism is involved.     

Verapamil enhances the efficiency of DNA-mediated gene transfer in mammalian cells

Treatment of Ltk^? cells with the calcium antagonists, verapamil and diltiazem, but not nifedipin, causes a 3-fold enhancement of the frequency of transfer of the cloned gene for herpes simplex virus thymidine kinase (HSV-tk). The frequency of phenotypic expression of the HSV-tk DNA was 20 to 34 times higher than that of genotypic transformation. Phenotypic expression was also 2.3 to 2.6 times increased when 20 μg/ml of verapamil was present during calcium phosphate-mediated DNA transfection.     

Human erythrocyte ammonium transport is mediated by functional interaction of ammonium (RhAG) and anion (AE1) transporters

The ammonia/ammonium (NH₃/NH ₄ ⁺ ) influx into red blood cells (RBCs) is mediated by surface glycoprotein RhAG that forms a structural complex with anion exchanger 1 (AE1, band 3). Owing to the activity of this complex, RBCs exposed to the isosmotic ammonium buffer swell and finally lyse. Isoosmotic NH ₄ ⁺ -containing media alters the pH gradient in RBCs (intracellular alkalosis in response to NH₃/NH ₄ ⁺ influx) and triggers the AE1 activity resulting in redundant chloride and water influx and finally in cell swelling. Here we demonstrate that the ammonia/ammonium transport in human RBCs depends on the pH (pH optimum 7.4 ± 0.1), temperature (Q₁₀ 2.6 ± 0.3), HCO ₃ ^? concentration (EC₅₀ 4.7 ± 0.3 mM), and AE1 function. The data confirm functional interactions between AE1 and RhAG. The initial velocity of cell swelling increased almost 50-fold in the isosmotic ammonium buffer containing 25 mM HCO ₃ ^? (37°C) in comparison to the reaction in the same buffer without HCO ₃ ^? . This indicates that the reaction is facilitated mostly by the carrier proteins, not just owing to the simple diffusion of NH₃ across the erythrocyte membrane. We demonstrate that pH_i reaches its maximum value much faster than the volume increase does. These data suggest that there is no direct correlation between pH_i changes and the influx of NH₃/NH ₄ ⁺ . Taken together, our data show that the RhAG and AE1 complex activity enables erythrocytes to be ammonia/ammonium storage sites in order to maintain the physiological blood ammonia/ammonium equilibrium.     

Reversal of phosphate induced decreases in force by the benzimidazole pyridazinone,UD-CG 212 CL,in myofilaments from human ventricle

UD-CG 212 Cl, (Fig. 1: 4,5-dihydro-6-[2-(4-hydroxyphenyl)-1H-benzimidazole-5-yl]-5-methyl-3(2H)-pyrid azinone), is the primary metabolite of the positive inotropic agent pimobendan (UDCG 115 BS, Acardi®). Our previous studies [16] showed in detergent extracted preparations of canine ventricular muscle that sub-nanomolar concentrations of UD-CG 212 Cl increased submaximal myofilament force, but only when the activation state had been altered by relatively high (5-10 mM) concentrations of inorganic phosphate (Pi) or relatively low (20 µM) concentrations of MgATP. In the present study, we investigated the effects of UD-CG 212 Cl on the pCa-force relationship of detergent extracted bundles of human cardiac fibers before and after addition of P_i. As expected, treatment with 5 mM P_i depressed maximal force at pCa 4.5 by 27.0 ± 0.4% (mean ± SEM). Force generated at the half-maximally activating Ca²⁺ concentration (pCa₅₀) of control fibers (5.98 ± 0.2) was significantly (p < .05) reduced following the addition of 5 mM P_i (pCa₅₀ = 5.69 ± 0.3). The addition of UD-CG 212 Cl over a range of concentrations (10-^-11>-10-^-6 M) had no effect on Ca²⁺-sensitivity under control conditions, but in the presence of 5 mM P_i, there was a 23.1 ± 0.1% increase in the percent maximal force at pCa5.9. Ca²⁺-sensitivity was also significantly increased in the presence of P_i and 10^-8 M UD-CG 212 Cl (pCa₅₀ = 5.74 ± 0.3, p < .05). We conclude that UD-CG 212 Cl potentially increases sub-maximal force of human ventricular myofilaments with an inotropic action depending on a state of myofilament activation associated with ischemic conditions.     

Role of Na+/ H+ exchange and HCO3 − transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen

This study sought to investigate effects of short-chain fatty acids and CO₂ on intracellular pH (pH_i) and mechanisms that mediate pH_i recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pH_i was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2′,7′-bis (carboxyethyl)-5(6′)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pH_i in N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 ± 0.03. In HEPES-buffered solution, a NH₄ ⁺/NH₃-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 μM) or HOE-694 (200 μM) inhibited pH_i recovery from an NH₄ ⁺/NH₃-induced acid load by 58% and 70%, respectively. pH_i recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 μM) and HOE-694 (200 μM), respectively. Changing from HEPES- (20 mM) to CO₂/HCO₃ ⁻-buffered (5%/20 mM) solution caused a rapid decrease of pH_i (P < 0.01), followed by an effective counter-regulation. 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 100 μM) blocked the pH_i recovery by 88%. The results indicate that intracellular acidification by butyrate and CO₂ is effectively counter-regulated by an Na⁺/H⁺ exchanger and by DIDS-sensitive, HCO₃ ⁻-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pH_i homeostasis of ruminal epithelial cells. Accepted: 8 March 2000