Ocean acidification and kelp development: Reduced pH has no negative effects on meiospore germination and gametophyte development of Macrocystis pyrifera and Undaria pinnatifida

The absorption of anthropogenic CO₂ by the oceans is causing a reduction in the pH of the surface waters termed ocean acidification (OA). This could have substantial effects on marine coastal environments where fleshy (non‐calcareous) macroalgae are dominant primary producers and ecosystem engineers. Few OA studies have focused on the early life stages of large macroalgae such as kelps. This study evaluated the effects of seawater pH on the ontogenic development of meiospores of the native kelp Macrocystis pyrifera and the invasive kelp Undaria pinnatifida, in south‐eastern New Zealand. Meiospores of both kelps were released into four seawater pH treatments (pH_T 7.20, extreme OA predicted for 2300; pH_T 7.65, OA predicted for 2100; pH_T 8.01, ambient pH; and pH_T 8.40, pre‐industrial pH) and cultured for 15 d. Meiospore germination, germling growth rate, and gametophyte size and sex ratio were monitored and measured. Exposure to reduced pH_T (7.20 and 7.65) had positive effects on germling growth rate and gametophyte size in both M. pyrifera and U. pinnatifida, whereas, higher pH_T (8.01 and 8.40) reduced the gametophyte size in both kelps. Sex ratio of gametophytes of both kelps was biased toward females under all pH_T treatments, except for U. pinnatifida at pH_T 7.65. Germling growth rate under OA was significantly higher in M. pyrifera compared to U. pinnatifida but gametophyte development was equal for both kelps under all seawater pH_T treatments, indicating that the microscopic stages of the native M. pyrifera and the invasive U. pinnatifida will respond similarly to OA.     

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO₃^?) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO₃^? by the surface‐bound enzyme carbonic anhydrase (CA_ext). Here, we examined other putative HCO₃^? uptake mechanisms in M. pyrifera under pH_T 9.00 (HCO₃^?: CO₂ = 940:1) and pH_T 7.65 (HCO₃^?: CO₂ = 51:1). Rates of photosynthesis, and internal CA (CA_int) and CA_ext activity were measured following the application of AZ which inhibits CA_ext, and DIDS which inhibits a different HCO₃^? uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO₃^? uptake by M. pyrifera is via an AE protein, regardless of the HCO₃^?: CO₂ ratio, with CA_ext making little contribution. Inhibiting the AE protein led to a 55%–65% decrease in photosynthetic rates. Inhibiting both the AE protein and CA_ext at pH_T 9.00 led to 80%–100% inhibition of photosynthesis, whereas at pH_T 7.65, passive CO₂ diffusion supported 33% of photosynthesis. CA_int was active at pH_T 7.65 and 9.00, and activity was always higher than CA_ext, because of its role in dehydrating HCO₃^? to supply CO₂ to RuBisCO. Interestingly, the main mechanism of HCO₃^? uptake in M. pyrifera was different than that in other Laminariales studied (CA_ext‐catalyzed reaction) and we suggest that species‐specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO₃^?:CO₂ due to ocean acidification.     

Apical and basolateral Na+/H+ exchange in the rabbit outer medullary thin descending limb of henle: Role in intracellular pH regulation

Summary The present study was designed to investigate the apical and basolateral transport processes responsible for intracellular pH regulation in the thin descending limb of Henle. Rabbit thin descending limbs of long-loop nephrons were perfused in vitro and intracellular pH (pH _i ) was measured using BCECF. Steady-state pH _i in HEPES buffered solutions (pH 7.4) was 7.18±0.03. Following the removal of luminal Na⁺, pH _i decreased at a rate of 1.96±0.37 pH/min. In the presence of luminal amiloride (1mm), the rate of decrease of pH _i was significantly less, 0.73±0.18 pH/min. Steady-state pH _i decreased 0.18 pH units following the addition of amiloride (1mm) to the lumen (Na⁺ 140mm lumen and bath). When Na⁺ was removed from the basolateral side of the tubule, pH _i decreased at a rate of 0.49±0.05 pH/min. The rate of decrease of pH _i was significantly less in the presence of 1mm basolateral amiloride, 0.29±0.04 pH/min. Addition of 1mm amiloride to the basolateral side (Na⁺ 140mm lumen and bath) caused steady-state pH _i to decrease significantly by 0.06 pH units. When pH _i was acutely decreased to 5.87±0.02 following NH₄Cl removal (lumen, bath), pH _i failed to recover in the absence of Na⁺ (lumen, bath). Addition of 140mm Na⁺ to the lumen caused pH _i to recover at a rate of 2.17±0.59 pH/min. The rate of pH _i recovery was inhibited 93% by 1mm luminal amiloride. When 140mm Na⁺ was added to the basolateral side, pH _i recovered only partially at 0.38±0.07 pH/min. Addition of 1mm basolateral amiloride inhibited the recovery of pH _i , by 97%. The results demonstrate that the rabbit thin descending limb of long-loop nephrons possesses apical and basolateral Na⁺/N⁺ antiporters. In the steady state, the rate of Na⁺-dependent H⁺ flux across the apical antiporter exceeds the rate of Na⁺-dependent H⁺ flux via the basolateral antiporter. Recovery of pH _i following acute intracellular acidification is Na⁺ dependent and mediated primarily by the luminal antiporter.     

Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger

We previously demonstrated that the progesterone‐ (P) initiated human sperm acrosome reaction (AR) was dependent on the presence of extracellular Na⁺ (Na⁺_o). Moreover, Na⁺_o depletion resulted in a decreased cytosolic pH (pH_i), suggesting involvement of a Na⁺‐dependent pH_i regulatory mechanism during the P‐initiated AR. We now report that the decreased pH_i resulting from Na⁺_o depletion is reversible and mediated by a Na⁺/H⁺ exchange (NHE) mechanism. To determine the role of an NHE in the regulation of pH_i, capacitated spermatozoa were incubated in Na⁺‐deficient, bicarbonate/CO₂‐buffered (0NaB) medium for 15–30 min, which resulted in an intracellular acidification as previously reported. These spermatozoa were then transferred to Na⁺‐containing, bicarbonate/CO₂‐buffered (NaB) medium; Na⁺‐containing, Hepes‐buffered (NaH) medium; or maintained in the 0NaB medium. Included in the NaH medium was the NHE inhibitor 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA). The steady‐state pH_i was then determined by spectrofluorometric measurement of bis(carboxyethyl)‐5(6)‐carboxyfluoroscein (BCECF) fluorescence. EIPA (0.1 μM) significantly (P < 0.05) inhibited the pH_i recovery produced by NaH medium. Moreover, the pH_i in NaH medium was not significantly (P < 0.05) different than NaB medium. These results indicate that a Na⁺‐dependent, bicarbonate‐independent pH_i regulatory mechanism, with a pharmacological characteristic consistent with an NHE, is present in capacitated spermatozoa. In support of the involvement of a sperm NHE, we also demonstrated specific immunoreactivity for a 100 kDa porcine sperm protein using an NHE‐1 specific monoclonal antibody. Interestingly, no significant (P = 0.79) effect was seen on the P‐initiated AR when EIPA was included in either the NaH or NaB medium. While these findings suggest that inhibition of NHE‐dependent pH_i regulation in capacitated spermatozoa is not sufficient to block initiation of the AR by P, they do not preclude the possibility that an NHE mediates the regulation of capacitation or sperm motility. Mol. Reprod. Dev. 52:189–195, 1999. © 1999 Wiley‐Liss, Inc.     

pH modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an outwardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M. 1985.J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistinguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examining F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was greater at lower (pH_int/pH_ext: 5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since stepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH (4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2 M). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at low pH (monovalent F^– predominates) and at least 2 H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparison of predictedvs. experimentally determined kinetic parameters at pH_ext5.8 (K _m =1.33vs. 1.70 M;V _max=123.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect theK _m for carrier-mediated F transport. These data are consistent with similarK _i ^' s for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 M, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalent F and is sensitive to external pH with a H⁺ K _m (or OH^– lC₅₀) corresponding to pH 4.89. External pH effects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺),rather than competitive binding that is mutually exclusive.     

Intracellular pH,intracellular free Ca,and junctional cell-cell coupling

Summary Intracellular pH (pH _i ) and intracellular Ca²⁺ ([Ca²⁺] _i ) were determined inChironomus salivary gland cells under various conditions of induced uncoupling. pH _i was measured with aThomas-type microelectrode, changes in [Ca²⁺] _i and their spatial distribution inside the cell were determined with the aid of intracellularly injected aequorin and an image intensifier-TV system, and cell-to-cell coupling was measured electrically. Treatments with NaCN (5mm), DNP (1.2mm), or ionophore A23187 (2m) caused fall in junctional conductance (uncoupling) that was correlated with [Ca²⁺] _i elevation, as was shown before (Rose & Loewenstein, 1976,J. Membrane Biol. 28:87) but not with changes in pH _i : during the uncoupling induced by CN, the pH _i (normally 7.5) decreased at most by 0.2 units; during the uncoupling induced by the ionophore, pH _i fell by 0.13 or rose by 0.3; and in any one of these three agents' uncouplings, the onset of uncoupling and recovery of coupling were out of phase with the changes in pH _i . Intracellular injection of Ca-citrate or Ca-EGTA solutions buffered to pH 7.2 or 7.5 produced uncoupling with little or no pH _i change when their free [Ca²⁺] _i was >10^–5 m. On the other hand, such a solution at pH 4, buffered to [Ca²⁺]<10^–6 m, lowered pH _i to 6.8 but produced no uncoupling. Thus, a decrease in pH _i is not necessary for uncoupling in any of these conditions. In fact, uncoupling ensued also during increase in pH _i : exposure to NH₄HCO₃ or withdrawal of propionate following exposure to a propionate-containing medium caused pH _i to rise to 8.74, accompanied by [Ca²⁺] _i elevation and uncoupling at pH _i >7.8.Cell acidification itself can cause elevation of [Ca²⁺] _i : injection (iontophoresis) of H⁺ invariably caused [Ca²⁺] _i elevation and uncoupling. These effects were produced also by an application of H⁺-transporting ionophore Nigericin at extracellular pH 6.5 which caused pH _i to fall to 6.8. Exposure to 100% CO₂ produced a fall in pH _i , associated in 10 out of 25 cases with [Ca²⁺] _i elevation and, invariably, with uncoupling. The absence of a demonstrable [Ca²⁺] _i elevation in a proportion of these trials is attributable to depression in Ca²⁺-measuring sensitivity; inin vivo tests, detection sensitivity for [Ca²⁺] _i by aequorin was found to be depressed by the CO₂ treatment. Upon CO₂ washout, pH _i and coupling recovered, but onset of recoupling set in at pH _i as low as 6.32–6.88, generally lower than at the pH _i at which uncoupling had set in. Exposure to 5% CO₂ lowered pH _i on the average by 0.3 and depressed coupling (in initially poorly coupled cells). After CO₂-washout, pH _i and coupling recovered. During the recovery phase [Ca²⁺] _i was elevated, an elevation associated with renewed uncoupling or decrease in rate of recoupling. The results are discussed in connection with possible regulatory mechanisms of junctional permeability.     

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.     

Mechanism of nitrate uptake into Chara corallina cells: lack of evidence for obligatory coupling to proton pump and a new NO3−/NO3− exchange model

Abstract. Nitrate uptake into Chara corallina cells at different external pH (pH_o) after different NO₃⁻ pretreatment conditions has been investigated. Following NO₃⁻ pretreatment (0.2 mol m⁻³ NO₃⁻) there was little effect of pH_o on subsequent net NO₃⁻ uptake into Chara cells. After N deprivation (2 mmol m⁻³ NO₃⁻) there was a pronounced effect of pH_o on nitrate uptake, the maximum rate occurring at pH_o 4.7. There was no consistent relationship between OH⁻ efflux and NO₃⁻ uptake in short term experiments (< 1 h). NO₃⁻ efflux was also sensitive to pH_o, the maximum rate occurring at ∼ pH_o 5.0. An inhibitor of the proton pump, DES, immediately stimulated NO₃⁻ uptake into cells pretreated with NO₃⁻ and prevented the time-dependent decrease in NO₃⁻, uptake into Chara cells that had been previously treated with low N (2 mmol m⁻³ NO₃⁻). NO₃⁻ efflux was found to be very sensitive to DES with K_i= 0.7 mmol m⁻³. At the optimum pH_o for NO₃⁻ uptake the effect of DES on membrane potential (ψ_m) were slight, and only apparent after 30 min. The results are interpreted in context of current models relating NO₃⁻ uptake and H⁺ pump activity. A new model for NO₃⁻ uptake is proposed which involves NO₃⁻/NO₃⁻ exchange at steady state.     

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl^? and HCO₃^? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO₃^? efflux through CFTR often required the intracellular H⁺/HCO₃^? production and/or the Na⁺-dependent basolateral HCO₃^? uptake, the intracellular pH (pH_i) balance might be disturbed, especially as a consequence of increased endogenous H⁺ and HCO₃^? production. However, measurement of pH_i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO₃^? transport. In addition, although the plasma membrane Na⁺/K⁺-ATPase (NKA) is normally essential for basolateral HCO₃^? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH_i or NKA abundance on cell membrane.     

Regulation of Intracellular pH in Guinea Pig Cerebral Cortex Ex Vivo Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy: Role of Extracellular Bicarbonate and Chloride

Abstract: The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH_i), high-energy metabolites, and lactate was investigated using ³¹P and ¹H NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO₃^?/CO₂ in the presence of Na⁺ led to a sustained split of the inorganic phosphate (P_i) peak so that the pH_i indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO₃^?. The pH in the compartment with a higher pH_i value returned to 7.29 ± 0.04 by 10.5 min of superfusion in a HCO₃^?-free medium, whereas the pH_i in an acidic compartment was reduced to 7.02. In the presence of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid or the absence of external Cl^?, removal of HCO₃^? caused alkalinization without split of the P_i peak. Both treatments reduced the rate of pH_i normalization following alkalinization. Simultaneous omission of external HCO₃^? and Na⁺ did not inhibit alkalinization of the pH_i following CO₂ exit. All these data show that the acid loading mechanism at neutral pH_i is mediated by an Na⁺-independent anion transport. During severe hypoxia, pH_i dropped from 7.29 ± 0.05 to 6.13 ± 0.16 and from 7.33 ± 0.03 to 6.67 ± 0.05 in the absence and presence of HCO₃^?, respectively, in Na⁺-containing medium. Lactate accumulated to 18.7 ± 2.8 and 19.6 ± 1.5 mmol/kg under the respective conditions. In the HCO₃^?-free medium supplemented with 1 mM amiloride, the pH_i fell only to 6.94 ± 0.08 despite the lactate concentration of 18.9 ± 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO₃^? and Cl^?, as the pH_i was 7.01 ± 0.12 at a lactate concentration of 24.5 ± 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO₃^? but <47% in those preparations superfused without HCO₃^? and Cl^?. This indicates that either neutral pH_i or absence of Cl^? during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl^?/HCO₃^? exchange mechanisms have distinct roles in cerebral H⁺ homeostasis depending on the level of pH_i and energy state.     

Effects on Mitochondrial K Flux of pH,K Concentration,and N-Ethyl Maleimide

Based on published evidence that cation transport in mitochondria is not significantly dependent on a membrane potential, it is suggested that the process of mitochondrial cation transport may be nonelectrogenic. These experiments focused on the possibility that K⁺ flux into rat liver mitochondria may be directly coupled, via an energy-linked carrier mechanism, to OH^? influx or H⁺ efflux. The dependence of the unidirectional K⁺ influx on the external K⁺ concentration indicates involvement of a saturable mechanism. Increasing the external pH from 7.0 to 8.0 increases the apparent V_max of the K⁺ influx without significantly altering the apparent K_m for K⁺. The pH dependence is greater in the presence of N-ethyl maleimide, a known inhibitor of the mitochondrial P_i/OH^? exchange mechanism. N-Ethyl maleimide decreases the apparent V_max at pH 7.0 and increases it at pH 8.0. Evidence indicates that both N-ethyl maleimide and a high external P_i concentration may stimulate the K⁺ influx at alkaline external pH (8.0) by preventing net exchanges between endogenous P_i and external OH^?. An apparent first-order dependence of the K⁺ influx on the external OH^? concentration is observed in the presence of N-ethyl maleimide. These results are consistent with a possible role of external OH^? as a cosubstrate of the K⁺ transport mechanism.     

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pH_i) and luminal pH (pH_lu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH_o 6.9). In proximal segments both pH_i (7.43±0.20) and pH_lu (7.10±0.24) were significantly lower than in distal segments (pH_i 7.70±0.29, pH_lu 8.09±0.15). Steady-state pH_i of proximal segments was much less sensitive to changes in pH_o than pH of the luminal fluid (pH_lu/pH_o was 0.49 while pH_i/pH_o was 0.18; pH_o 6.50–7.20). Re-alkaliniziation from an NH₄Cl-induced intracellular acid load (initial pH_i recovery rate 0.55±0.34 pH·min^-1) was nearly totally inhibited by 1 mmol·l^-1 KCN (96% inhibition) and to a large degree (79%) by 1 mol·l^-1 bafilomycin A₁. In contrast, both vanadate (1 mmol·l^-1) and amiloride (1 mmol·l^-1) inhibited pH_i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na⁺ from the bathing saline had no effect on pH_i recovery, indicating that a Na⁺/H⁺ exchange is not significantly involved in pH_i regulation. Instead pH_i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.Abbreviations DMSO dimethylsulphoxide - DNP 2,4-dinitrophenol - NMDG N-methyl-D-glucamine - pH_i intracellular pH - pH_lu pH of the luminal fluid - pH_o pH of the superfusion medium - _I intrinsic intracellular buffer capacity     

Intracellular pH change does not accompany egg activation in the mouse

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pH_i). We measured pH_i, in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH_i was 6.96 ± 0.004 (± SEM) in GV-intact oocytes, 7.00 ± 0.01 in ovulated, unfertilized eggs, and 7.02 ± 0.01 in fertilized zygotes, indicating no sustained changes in pH_i after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pH_i occur shortly after egg activation, mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS); no significant change in pH_i followed ethanol activation. Since increased Na⁺/H⁺ antiporter activity is responsible for pH_i increase in the sea urchin, pH_i was measured in the absence of added bicarbonate or CO₂ la condition under which the antiporter would be the only major pH_i regulatory mechanism able to operate, since the others were bicarbonate- dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na⁺/H⁺ antiporter was activated. There was no physiologically significant difference in pH_i after GVBD or fertilization, when pH_i was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation. Thus, a change in pH_i is not a feature of egg activation in the mouse. © 1996 Wiley-Liss, Inc.     

Effects of pH and nitrogen on cadmium uptake in potato

This study investigated the effects of pH and nitrogen form and concentration on cadmium (Cd) uptake by potato (Solanum tuberosum L.) grown in hydroponic culture. Potato plants grown in a pH-buffered nutrient solution for 10 d were exposed for 24 h to 25 nM CdCl₂ labelled with ¹⁰⁹Cd. Plants showed a significantly higher Cd uptake and accumulation at pH 6.5 than at pH 4.5 and 5.5. Nitrogen supplied as nitrate (NO₃ ^?) generally resulted in a higher Cd uptake and accumulation than N supplied as ammonium (NH₄ ⁺). This effect was most pronounced at pH 6.5. The N concentration increasing from 6.5 to 26 mM resulted in a decreased Cd influx when either NO₃ ^? or NH₄ ⁺ was used. Cd translocation to the shoot was increased when NO₃ ^? was used as the sole N source. In conclusion, pH had a strong influence on Cd uptake by roots and N form is especially important for Cd translocation within the potato plant.     

Amiloride-sensitive Na+ transport in human red cells: Evidence for a Na/H exchange system

Summary The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na⁺ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 m) and methazolamide (400 m). Red cells with different internal pH (pH _i =6.4, 7.0 and 7.8) were prepared and Na⁺ influx was measured at different external pH (pH _o =6.0, 7.0, 8.0). Na⁺ influx into acid-loaded cells (pH _i =6.4) markedly increased when pH _o was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na⁺/H⁺ exchange systems blocked about 60% of the H⁺-induced Na⁺ entry, while showing small inhibitory effects in the absence of pH gradients. When pH₀ was kept at 8.0, the amiloride-sensitive Na⁺ entry was abolished as pH _i was increased from 6.4 to 7.8. Moreover, measurements of H⁺ efflux into lightly buffered media indicated that the imposition of an inward Na⁺ gradient stimulated a net H⁺ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na⁺ (Na _i ⁺ =Na ₀ ⁺ =15mm,Em=+6.7 mV), an outward H⁺ gradient (pH _i =6.4, pH₀=8.0) promoted a net amiloride-sensitive Na⁺ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na⁺/H⁺ exchange system in human red cells.     

Intracellular pH Increase Driven by an Na+/H+ Exchanger upon Activation of Surf Clam Oocytes

Intracellular pH (pH_i) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pH_iof 0.3 pH unit after activation by excess K⁺. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pH_iwas increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K⁺induced GVBD but the pH_iof K⁺-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pH_iincreases in K⁺-activated oocytes were otherwise proportional to the external Na⁺concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10–50 μM) efficiently inhibited the K⁺-induced increase of pH_ibut did not block GVBD. These two derivatives were able, however, to retard K⁺-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K⁺-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pH_iincrease, driven by a typical Na⁺/H⁺exchanger, follows activation of surf clam oocytes but that this pH_iincrease is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.     

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.     

FLOW‐INDUCED MORPHOLOGICAL VARIATIONS AFFECT DIFFUSION BOUNDARY‐LAYER THICKNESS OF MACROCYSTIS PYRIFERA (HETEROKONTOPHYTA,LAMINARIALES)1

In slow mainstream flows (<4–6 cm · s^?1), the transport of dissolved nutrients to seaweed blade surfaces is reduced due to the formation of thicker diffusion boundary layers (DBLs). The blade morphology of Macrocystis pyrifera (L.) C. Agardh varies with the hydrodynamic environment in which it grows; wave‐exposed blades are narrow and thick with small surface corrugations (1 mm tall), whereas wave‐sheltered blades are wider and thinner with large (2–5 cm) edge undulations. Within the surface corrugations of wave‐exposed blades, the DBL thickness, measured using an O₂ micro‐optode, ranged from 0.67 to 0.80 mm and did not vary with mainstream velocities between 0.8 and 4.5 cm · s^?1. At the corrugation apex, DBL thickness decreased with increasing seawater velocity, from 0.4 mm at 0.8 cm · s^?1 to being undetectable at 4.5 cm · s^?1. Results show how the wave‐exposed blades trap fluid within the corrugations at their surface. For wave‐sheltered blades at 0.8 cm · s^?1, a DBL thickness of 0.73 ± 0.31 mm within the edge undulation was 10‐fold greater than at the undulation apex, while at 2.1 cm · s^?1, DBL thicknesses were similar at <0.07 mm. Relative turbulence intensity was measured using an acoustic Doppler velocimeter (ADV), and overall, there was little evidence to support our hypothesis that the edge undulations of wave‐sheltered blades increased turbulence intensity compared to wave‐exposed blades. We discuss the positive and negative effects of thick DBLs at seaweed surfaces.     

AMMONIUN AND NITRATE UPTAKE BY THE MARINE MACROPHYTES HYPNEA MUSVUFORMIS (RHODOPHYTA) AND MACROCYSTIS PYRIFERA (PHAEOPHYTA)1, 2

NH₄⁺ and NO₃^? uptake were measured by continuous sampling with an autoanalyzer. For Hypnea musciformis (Wulfen) Lamouroux, NO₃^?up take followed saturable kinetics (K₂=4.9 μg-at N t^?1, V_max= 2.85 μg- at N, g(wet)^?1. h^?1. The ammonium uptake data fit a trucatd hyperbola, i.e., saturation was not reach at the concentrations used. NO₃^? uptake was reduced one-half in the presence of NH₄⁺, but presence of NO₃^? had no effect on NH₄⁺ uptake. Darkness reduced both NO₃^? and NH₄⁺ uptake by one-third to one-half. For Macrocystis pyrufera (L) C. Agardh, NO₃^? uptake followed saturable kinetices: K₂=13.1 μg-at N. l^?1. V_max=3.05 μg-at N. g(wet)^?1. h^?1.NH₄⁺ uptake showed saturable kinetics at concentration below 22 μg-at N l -1 (K₂=5.3 μg-at N.1–1, V_max= 2.38 μg-at N G (wet)^?1.h^?1: at higher concentration uptake increased lincarly with concentrations. NO₃^?and NH₄⁺ were taken up simulataneously: presence of one form did not affect uptake of the other.