Intracellular pH regulation in human preimplantation embryos

We report here that intracellular pH (pH(i)) in cleavage-stage human embryos (2-8-cell) is regulated by at least two mechanisms: the HCO(3)(-)/Cl(-) exchanger (relieves alkalosis) and the Na(+)/H(+) antiporter (relieves acidosis). The mean pH(i) of cleavage-stage embryos was 7.12 +/- 0.008 (n = 199) with little variation between different stages. Embryos demonstrated robust recovery from alkalosis that was appropriately Cl(-)-dependent, indicating the presence of the HCO(3)(-)/Cl(-) exchanger. This was further confirmed by measuring the rate of intracellular alkalinization upon Cl(-) removal, which was markedly inhibited by the anion transport inhibitor, 4,4'-diisocyanatostilbene-2,2'-disulphonic acid, disodium salt. The set-point of the HCO(3)(-)/Cl(-) exchanger was between pH(i) 7.2 and 7.3. Embryos also exhibited Na(+)-dependent recovery from intracellular acidosis. Na(+)/H(+) antiporter activity appeared to regulate recovery up to about pH(i) 6.8; this recovery was HCO(3)(-)-independent and amiloride-sensitive, with a pH(i) set-point of approximately 6.8-6.9. A second system that was both Na(+)- and HCO(3)(-)-dependent appeared to mediate further recovery from acidosis up to about pH(i) 7.1. Thus, pH(i) of early human preimplantation embryos appears to be regulated by opposing mechanisms (HCO(3)(-)/Cl(-) exchanger, Na(+)/H(+) antiporter, and possibly a third acid-alleviating transporter that was both Na(+)- and HCO(3)(-)-dependent) resulting in the maintenance of pH(i) within a narrow range.     

Cryopreservation reduces the ability of hamster 2-cell embryos to regulate intracellular pH

Vitrification of hamster 2-cell embryos impairs the activity of both the Na(+)/H(+) antiporter and HCO(3)(-)/Cl(-) exchanger; the two transport proteins responsible for the regulation of intracellular pH (pHi). The activities of both the Na(+)/H(+) antiporter and HCO(3)(-)/Cl(-) exchanger were significantly reduced at 4 h following warming compared to freshly collected embryos. Normal levels of activity of both transporters were not restored until 6 h after warming. Thus, cryopreservation of cleavage stage hamster embryos has a detrimental effect on their ability to maintain intracellular ionic homeostasis. Impairment of these pHi regulatory proteins resulted in the pHi of embryos being significantly elevated from the control values of 1.2 to 7.35 for approximately 4 h after warming. In addition, an elevated pHi value significantly impaired oxidative metabolism. Therefore, the loss in developmental competence of embryos following cryopreservation may in part be explained by a reduced ability to regulate intracellular pH that results in perturbations in metabolism and disruption of energy production.     

Effects of temperature on intracellular sodium, pH and cellular energy status in RIF-1 tumor cells

Most perfused tumor cell experiments are performed at 37 degrees C, the normal healthy body temperature. However, the temperature of subcutaneously implanted tumors in small animals is generally 29-33 degrees C when the rectal temperature of the animal is maintained at 37 degrees C. We have investigated the acute effects of increasing the temperature of perfused radiation-induced-fibrosarcoma (RIF-1) tumor cells from 33 to 37 degrees C (30 min) on intracellular sodium (Na(i)+) , intracellular pH (pH(i)), and bioenergetic status. Heating the cells by 4 degrees C produced a reversible increase in Na(i)+, slight acidification and no change in nucleotide triphosphate to inorganic phosphate ratio (NTP/P(i)) as measured by shift-reagent-aided (23)Na and (31)P NMR spectroscopy. In the presence of 3 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a potent and specific inhibitor of Na(+)/H(+) antiporter, the increase in Na(i)+ during the heating was completely abolished suggesting that the heat induced increase in Na(i)+ was caused by an increase in Na(+)/H(+) antiporter activity. However, the changes in pH(i) with the heating were identical with or without EIPA, indicating that pH(i) is controlled by other ion exchange mechanisms in addition to Na(+)/H(+) antiporter. NTP/P(i) was significantly higher in presence of EIPA for some time points during the heating suggesting that both NTP production and consumption rates may be altered during the heating. These results indicate that a slight increase in temperature from 33 to 37 degrees C induces significant changes in Na(+) physiology largely because of activation of Na(+)/H(+) antiporter but other ion exchange mechanisms are also involved in maintaining pH(i) in the RIF-1 tumor cells. Thus, care must be taken in choosing the temperature for perfused cell studies.     

Acid extrusion from the intraerythrocytic malaria parasite is not via a Na(+)/H(+) exchanger

The intraerythrocytic malaria parasite, Plasmodium falciparum maintains an intracellular pH (pH(i)) of around 7.3. If subjected to an experimentally imposed acidification the parasite extrudes H(+), thereby undergoing a pH(i) recovery. In a recent study, Bennett et al. [Bennett TN, Patel J, Ferdig MT, Roepe PD. P. falciparum Na(+)/H(+) exchanger activity and quinine resistance. Mol Biochem Parasitol 2007;153:48-58] used the H(+) ionophore nigericin, in conjunction with an acidic medium, to acidify the parasite cytosol, and then used bovine serum albumin (BSA) to scavenge the nigericin from the parasite membrane. The ensuing Na(+)-dependent pH(i) recovery, seen following an increase in the extracellular pH, was attributed to a plasma membrane Na(+)/H(+) exchanger. This is at odds with previous reports that the primary H(+) extrusion mechanism in the parasite is a plasma membrane V-type H(+)-ATPase. Here we present evidence that the Na(+)-dependent efflux of H(+) from parasites acidified using nigericin/BSA is attributable to Na(+)/H(+) exchange via residual nigericin remaining in the parasite plasma membrane, rather than to endogenous transporter activity.     

Na+/H+ antiporter has different properties in human B lymphocytes according to CD5 expression and malignant phenotype.

In B chronic lymphocytic leukemia (B-CLL) cells, lipopolysaccharide (LPS) and phorbol esters fail to activate the plasma membrane-associated Na+/H+ antiporter and, subsequently, to elicit a rise in cytosolic pH. Since these events are thought to be a prerequisite for LPS-induced proliferation of B normal lymphocytes, we analyzed the kinetic properties of Na+/H+ antiporter in B-CLL cells as compared to both CD5- and CD5+ normal B lymphocytes. In the present work we report that Na+/H+ exchange rate after acid loading is drastically decreased in B-CLL cells, as compared to normal CD5- B lymphocytes, although the antiporter affinity for external Na+ and internal H+ is not significantly different in both cell populations. Kinetic data account for a reduction in the number of operating antiport units in B-CLL. The Na+/H+ antiporter of CD5+ normal B lymphocytes exhibits both an exchange rate and an ion affinity significantly higher than that observed in both CD5+ B-CLL cells and CD5- B normal lymphocytes, thus suggesting a possible explanation for their activated phenotype.     

The role of HCO3(-)-dependent mechanisms in pHi regulation during O2 deprivation

We have reported in our previous work that, in the absence of HCO(3)(-), Na(+)/H(+) exchanger is responsible for an anoxia-induced alkalinization in hippocampal CA1 neurons. HCO(3)(-)-dependent mechanisms have been reported to play a key role in pH(i) regulation in nerve cells, but how their function is affected by O(2) deprivation has not been well studied. In this work, pH(i) measurements (obtained from dissociated neurons loaded with carboxy-seminaphthorhodafluor-1 and using confocal microscopy) and whole-cell patch clamp recording techniques were used to investigate the role of HCO(3)(-)-dependent membrane exchangers on CA1 neurons during O(2) deprivation. Anoxia (5 min) induced a small acidification in neurons in the presence of HCO(3)(-) and this acidification was changed to a significant alkalinization when neurons were bathed with Hepes buffer or when 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was applied in a HCO(3)(-) solution, indicating that HCO(3)(-)-dependent mechanisms were involved. A marked anoxia-induced acidification (0.33+/-0.11 pH unit) was seen when the Na(+)/H(+) exchange was blocked with 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate in the presence of HCO(3)(-), but the same anoxia did not cause a significant pH(i) change in a Na(+) free, HCO(3)(-) solution, suggesting that the anoxia-induced acidification in the presence of 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate is dependent on both Na(+) and HCO(3)(-). Furthermore, anoxia did not cause a significant pH(i) change when both 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate were present. Current clamp recordings showed a significant membrane depolarization following anoxia in HCO(3)(-) solution but not in Hepes buffer. Our data suggest that, in hippocampal neurons: a) pH(i) regulation during O(2) deprivation is affected not only by metabolism but also by membrane exchangers, and b) besides the activation of Na(+)/H(+) exchange, anoxia activates a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive, Na(+)-dependent acid loader (possibly electrogenic).     

A plasma membrane P-type H(+)-ATPase regulates intracellular pH in Leishmania mexicana amazonensis

A recent report (Mukherjee et al., J. Biol. Chem. 276 (2001) 5563) has proposed that the plasma membrane Mg(+)-ATPase of promastigotes of Leishmania donovani, that is involved in its intracellular pH regulation, is an electroneutral H(+)/K(+) antiporter rather than an electrogenic H(+) pump. Since this proposition has important implications for the use of the pump as a target for chemotherapy, we investigated its nature in the mammalian stage (amastigote) of L. mexicana amazonensis and compared it with that present in promastigotes. Intracellular pH and H(+) efflux were measured using the acetotoxymethyl ester and the free form of 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein, respectively. Intracellular pH in amastigotes (at an external pH of 5.5) and promastigotes (at an external pH of 7.4) was 6.36+/-0.02 and 6.83+/-0.07, respectively. Differences in the mechanisms for regulation of intracellular pH were noted between amastigote and promastigote forms. Amastigotes maintained their intracellular pH neutral over a wide range of external pHs in the absence of K(+) or Na(+). The H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide, diethylstilbestrol and N-ethylmaleimide, substantially decreased their steady-state intracellular pH, inhibited proton efflux, and their recovery from acidification. The data support the presence of an H(+)-ATPase as the major regulator of intracellular pH in amastigotes. In contrast, promastigotes were unable to maintain a neutral pH under acidic conditions and although their steady-state intracellular pH and recovery from acidification were affected by H(+)-ATPase inhibitors, bicarbonate was able to overcome intracellular acidification. Bicarbonate was also able to raise the steady-state intracellular pH from 6.80+/-0.03 to 7.25+/-0.09 and induce membrane hyperpolarization. No evidence was found of the possible involvement of a K(+)/H(+)-ATPase in intracellular pH regulation in both developmental stages of L. m. amazonensis.     

Elevated rate of Thelper1 (T(H)1) lymphocytes and serum IFN-gamma levels in psoriatic patients

Several disorders are known to be associated with altered Thelper1/Thelper2 (T(H)1/T(H)2) cytokine balance. Psoriasis is characterized by increased systemic and local production of T(H)1 and pro-inflammatory cytokines. Furthermore recent data indicate the dominant presence of T(H)1 lymphocytes in the circulation and T(H)1 and Tcytotoxic1 (T(C)1) cells in lesional skin of psoriatic patients. In order to assess the systemic T(H)1/T(H)2 imbalance in psoriasis most of the studies so far tested isolated peripheral mononuclear cells. As a new approach we applied a whole blood flow cytometric assay to determine the rate of circulating T(H)1/T(H)2 and T(C)1/Tcytotoxic2 (T(C)2) lymphocytes based on their intracellular IFN-gamma, IL-4 and IL-10 expression. Besides, serum levels of these cytokines were determined in healthy controls and psoriatic patients by commercial ELISAs. In psoriatic patients we found significantly (P<0.02) increased rates of CD4(+)/IFN-gamma(+) lymphocytes (30.3+/-8.8%) while the percent of CD4(+)/IL-4(+) cells (0.37+/-0.31%) were significantly (P<0.03) lower compared to healthy controls (CD4(+)/IFN-gamma(+): 20.1+/-7.3% and CD4(+)/IL-4(+): 0.78+/-0.44%). The IL-10-positive CD4(+) and CD8(+) cells also had higher rate in psoriasis, but the difference between patients and controls was not significant, similarly to the rate of CD8(+)/IFN-gamma(+) and CD8(+)/IL-4(+) lymphocytes. Beside cellular expression, serum IFN-gamma levels were also significantly higher (control: 4.9+/-6.4 pg/ml; psoriatic patients: 35.9+/-47.0 pg/ml; P<0.05). Our results provide further evidence for an altered T(H)1/T(H)2 balance in psoriasis measured in non-separated whole blood T cells.     

Na(+)-H+ exchange is not important for pancreatic HCO3- secretion in the pig.

Pancreatic inter- and intralobular duct cells extrude H(+)-ions to interstitial fluid when they secrete HCO3- to pancreatic juice. This study assesses the potential importance of Na(+)-H(+)-ion exchange for H(+)-ion extrusion and secretion of HCO3-, using the Na(+)-H+ exchange blockers amiloride and hexamethylene-amiloride. Intracellular pH (pHi) in inter- and intralobular pancreatic duct epithelium was measured using BCECF fluorescence. H(+)-ion efflux was measured using a NH4Cl prepulse, acid-loading technique. In HCO3(-)-free media, pHi recovery following acid loading was blocked by amiloride (10(-4) M) and hexamethylene-amiloride (10(-6) M), demonstrating amiloride- and hexamethylene-amiloride-sensitive Na(+)-H+ exchange. However, 5 x 10(-6) M hexamethylene-amiloride did not reduce secretin-dependent pancreatic HCO3- secretion in vivo. Maximal H(+)-efflux through Na(+)-H+ exchange was 1.5 +/- 0.2 mumol min-1 ml cell volume-1, i.e. less than 1% of estimated net H(+)-ion efflux during HCO3- secretion. Conclusion: amiloride- and hexamethylene amiloride sensitive Na(+)-H+ exchange is not important for secretin-dependent pancreatic HCO3- secretion in the pig. Other mechanisms for H+ extrusion dominate.     

Role of Na+/Ca2+ exchange in [Ca2+]i clearance in rat culture Purkinje neurons requires reevaluation

Previous studies have shown that in contrast to other neuronal cells, Na(+)/Ca(2+) exchange contributes little to Ca(i)(2+) homeostasis in rat cerebellar Purkinje neurons under intracellular perfused conditions and at room temperature [Fierro et al.: J Physiol (Lond) 510: 499-512, 1998]. The purpose of this study was to clarify the role of this transporter in cerebellar Purkinje neurons by using intact cells at nearly physiological body temperature. Using Fluo-3 microfluorometry, we have examined the role of the Na(+)/Ca(2+) exchange in the buffering of calcium loads in cultured rat Purkinje neurons at two temperatures: 20 and 34 degrees C. At 20 degrees C, the recovery of the K(+)-induced [Ca(2+)](i) signal was little affected by the presence of external Na(+) (tau(e) = 35.5 +/- 1.2 s [n = 49]), or by its absence (tau(e) = 36.6 +/- 2.2 s [n = 29]), i.e. in a Li(+)-containing medium. In contrast, at 34 degrees C, the recovery of the [Ca(2+)](i) signal was highly dependent on external Na, i.e. tau(e) = 19.9 +/- 1.2 s (n = 119) and tau(e) = 41.7 +/- 2.6 s (n = 39), in Li(+)-containing media, respectively. A comparison of the rate of clearance of [Ca(2+)](i) in Na(+) or Li(+) media, shows that at a room temperature of 20 degrees C, the Na(+)/Ca(2+) exchange contributes at most to 15-20% of the total [Ca(2+)](i) clearance, compared to 55-65% at 34-36 degrees C. We also demonstrate that under normal physiological conditions forward and reverse Na(+)/Ca(2+) exchanges operate in the same neuron. We conclude that the Na(+)/Ca(2+) exchange is strongly suppressed at room temperature and therefore its role should be reevaluated among different neuronal preparations.     

Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2

We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na(+)/H(+) antiport were determined fluorometrically; the rates of Na(+)-K(+)-2Cl(-) symport and Na(+)/K(+)-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive (86)Rb(+) uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd(3+), flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na(+) over K(+). Hypertonic stress did not change K(+) conductance, whereas possible changes in Cl(-) conductance remain ambiguous. The contribution of Na(+)/H(+)antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na(+)-K(+)-2Cl(-)symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na(+)/K(+)-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K(+) conductance, Na(+)/H(+) antiport, Na(+)-K(+)-2Cl(-) symport, and Na(+)/K(+)-ATPase to this process.     

Intracellular pH response to anoxia in acutely dissociated adult rat hippocampal CA1 neurons

The effects of anoxia on intracellular pH (pH(i)) were examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H(+)-sensitive fluorophore, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. During perfusion with HCO/CO(2)- or HEPES-buffered media (pH 7.35) at 37 degrees C, 5- or 10-min anoxic insults were typified by an intracellular acidification on the induction of anoxia, a subsequent rise in pH(i) in the continued absence of O(2), and a further internal alkalinization on the return to normoxia. The steady-state pH(i) changes were not consequent on changes in [Ca(2+)](i) and, examined in the presence of HCO, were not significantly affected by (DIDS). In the absence of HCO, the magnitude of the postanoxic alkalinization was attenuated when external Na(+) was reduced by substitution with N-methyl-D-glucamine (NMDG(+)), but not Li(+), suggesting that increased Na(+)/H(+) exchange activity contributes to this phase of the pH(i) response. In contrast, 100-500 microM Zn(2+), a known blocker of H(+)-conductive pathways, reduced the magnitudes of the internal alkalinizations that occurred both during and following anoxia. The effects of NMDG(+)-substituted medium and Zn(2+) to reduce the increase in pH(i) that occurred after anoxia were additive. Consistent with the steady-state pH(i) changes, rates of pH(i) recovery from internal acid loads imposed immediately after anoxia were increased, and the application of Zn(2+) and/or perfusion with NMDG(+)-substituted medium slowed pH(i) recovery. Reducing extracellular pH from 7.35 to 6.60, or reducing ambient temperature from 37 degrees C to room temperature, also attenuated the increases in steady-state pH(i) observed during and after anoxia and reduced rates of pH(i) recovery from acid loads imposed in the immediate postanoxic period. Finally, inhibition of the cAMP/protein kinase A second-messenger system reduced the magnitude of the rise in pH(i) after anoxia in a manner that was dependent on external Na(+); conversely, activation of the system with isoproterenol increased the postanoxic alkalinization, an effect that was attenuated by pretreatment with propranolol, Rp-cAMPS, or when NMDG(+) (but not Li(+)) was employed as an external Na(+) substitute. The results suggest that a Zn(2+)-sensitive acid efflux mechanism, possibly a H(+)-conductive pathway activated by membrane depolarization, contributes to the internal alkalinization observed during anoxia in adult rat CA1 neurons. The rise in pH(i) after anoxia reflects acid extrusion via the H(+)-conductive pathway and also Na(+)/H(+) exchange, activation of the latter being mediated, at least in part, through a cAMP-dependent signaling pathway.     

Ion regulation in the different life stages of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>

Different ion and pH regulation mechanisms have been detected in the three main life stages of Trypanosoma cruzi: epimastigote, metacyclic trypomastigote and amastigote. Treatment with amiloride showed that the Na(+)/H(+) exchanger participated in all three forms. The Na(+)/K(+) ATPase exchanger appeared to be more active in the epimastigote than in the other forms. V-H(+)-ATPase inhibitors revealed the activity of this regulatory mechanism in the amastigote and epimastigote forms, while treatment with oligomycin only affected the amastigotes. The HCO(-)(3)/Cl(-) exchanger was found in all stages as well as in the intracellular pH-regulatory mechanism after abrupt basification. We deduce that ion regulation in T. cruzi is a complex process and depends upon the precise stage of the cell cycle of the parasite. It would seem to be an important mechanism, allowing the parasite to adapt to the changing environmental conditions within which it develops.     

The role of the Na+/H+ antiporter in human neutrophil NADPH-oxidase activation

The Na+/H+ antiporter has been shown to regulate activation of the human neutrophil. To delineate the role of the antiporter in the stimulation cascade, superoxide [O2-) generation was observed in PMA-stimulated neutrophils in which intracellular pH (pHi) was artificially manipulated by the use of nigericin, a K+/H+ ionophore, with and without use of the Na+/H+ antiporter inhibitor, dimethylamiloride (DA). Decreased O2- generation was observed in a Na+-free, 140 mM K+ buffer, but addition of nigericin restored this parameter of neutrophil activation to levels found in physiologic Na+ media. Further, the inhibitory effects of DA on O2- generation by cells incubated in a 10 mM Na+, 130 mM K+ buffer were totally reversed by a similar concentration of nigericin. O2- generated by a membrane preparation of the NADPH-oxidase, made from cells incubated under the same experimental conditions, paralleled whole cell studies, but the activity of the oxidase did not vary when suspended in the various reaction mixtures. These experiments support the role of the Na+/H+ antiporter in neutrophil activation as a metabolic regulator of pHi that influences receptor-coupled reactions proximal to expression of the NADPH-oxidase itself.     

Optimal absorptive transport of the dipeptide glycylsarcosine is dependent on functional Na+/H+ exchange activity

Optimal nutrient absorption across the intestinal epithelium is dependent on the co-ordinated activity of a number of membrane transporters. Di/tripeptide transport across the luminal membrane of the intestinal enterocyte is mediated by the H(+)-coupled di/tripeptide transporter hPepT1. hPepT1 function is dependent on the existence of a pH gradient (maintained, in part, by the action of the Na(+)/H(+) exchanger NHE3) across the apical membrane of the small intestinal epithelium. The physiological problem addressed here was to determine how two transporters (hPepT1 and NHE3), involved in nutrient absorption and pH(i) homeostasis, function co-operatively to maximise dipeptide absorption when both operate sub-optimally at typical mucosal surface pH values (pH 6.1-6.8). Functional hPepT1 activity in human intestinal epithelial (Caco-2) cell monolayers was determined by measurement of apical uptake and apical-to-basolateral transport of the dipeptide glycylsarcosine. The dependence of hPepT1 on NHE3 activity was measured (either after Na(+) removal or addition of the NHE3-selective inhibitor S1611) using both Caco-2 cell monolayers and hPepT1-expressing Xenopus laevis oocytes. Apical glycylsarcosine uptake in Caco-2 cell monolayers was modulated by apical pH, extracellular Na(+), incubation time and S1611. Uptake in hPepT1-expressing oocytes was independent of Na(+) or S1611. We conclude that functional NHE3 activity is required to allow optimal absorption of dipeptides across the human intestinal epithelium.     

The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis

Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.     

Transcellular transport of isosmotic volumes by the rabbit gall-bladder in vitro

1. Fluid transport rate and oxygen consumption (Q(O2)) were studied in rabbit gall-bladder preparations in vitro exposed on both sides to identical Ringer solutions with NaCl concentrations (and osmolarities) varying from 70 to 140 m-equiv Na(+)/l.).2. The time sequence of acute effects on transport rate resulting from sudden changes in the NaCl concentration of the bathing solutions indicated that, (a) as a primary effect, fluid volume transfer rate remained unaffected whereas Na transport rate changed abruptly in direct proportion to the Na concentration of the bathing media; (b) a secondary, delayed and partly reversible depression of fluid transfer rate following elevation of the NaCl concentration was observed only when the rate of transport was relatively high initially.3. A fixed, and highly significant, linear relationship between changes in transport-linked oxygen consumption (DeltaQ(O2)) and measured net fluid volume transport (DeltaT(vol)) was found independent of the NaCl concentration of the bathing media, dQ(O2)/dT(vol) being 0.22 +/- 11% and 0.25 +/- 8% in bladders incubated in solutions containing 140 and 70 m-equiv Na(+)/l. respectively.4. Oxygen consumption per equiv of Na(+) (calculated) transported varied in inverse proportion to the Na concentration of the bathing media, dQ(O2)/dT(Na) being 0.0016 +/- 11% and 0.0036 +/- 8% in ;140 R' and ;70 R' solutions, respectively.5. Removal of K from the bathing solutions was followed by a gradual and partly reversible depression of fluid transport rate to a minimum level (about 100 x 10(-4) mul H(2)O. min(-1).mg(-1)) independent of the initial transport rate.6. It is concluded that the range of absorption rates of isosmotic fluid from the gall-bladder lumen represents a range of energy requiring capacities for transfer of fluid volume units; the data suggest that the intracellular (cytoplasmic) ion composition, depending on the presence of external K, as well as hormonal action may influence the capacity of the transcellular fluid transport mechanism.7. A model (a ;mechanical volume pump') for transcellular transfer of fluid volume units, allowing for flexible specificity with regard to the actively transported solutes, and requiring the presence of Na(+) and Cl(-), is proposed.     

Differences in relationships among sleep apnoea, glucose level, sleep duration and sleepiness between persons with and without type 2 diabetes

Obstructive sleep apnoea is common in patients with diabetes. Recently, it was reported that short sleep duration and sleepiness had deleterious effects on glucose metabolism. Thereafter, several reports showed relationships between glucose metabolism and obstructive sleep apnoea, sleep duration or sleepiness. But the interrelationships among those factors based on recent epidemiological data have not been examined. We analysed data on 275 male employees (age, 44±8years; body mass index, 23.9±3.1kg m(-2) ) who underwent a cross-sectional health examination in Japan. We measured fasting plasma glucose, sleep duration using a sleep diary and an actigraph for 7days, and respiratory disturbance index with a type 3 portable monitor for two nights. Fifty-four subjects (19.6%) had impaired glucose metabolism, with 21 having diabetes. Of those 21 (body mass index, 25.9±3.8kgm(-2) ), 17 (81.0%) had obstructive sleep apnoea (respiratory disturbance index≥5). Regarding the severity of obstructive sleep apnoea, 10, four and three had mild, moderate and severe obstructive sleep apnoea, respectively. The prevalence of obstructive sleep apnoea was greater in those with than without diabetes (P=0.037). Multiple regression analyses showed that the respiratory disturbance index independently related to fasting plasma glucose only in the diabetic subjects. In patients with diabetes, after adjustment for age, waist circumference, etc. sleep fragmentation had a greater correlation with fasting plasma glucose than sleep duration, but without significance (P=0.10). Because the prevalence of obstructive sleep apnoea is extremely high in patients with diabetes, sufficient sleep duration with treatment for obstructive sleep apnoea, which ameliorates sleep fragmentation, might improve fasting plasma glucose.     

Alkaline pH changes in the cerebellum of asymptomatic HIV-infected individuals

Human immunodeficiency virus (HIV) infection of the brain causes a complex cascade of cellular events involving several different cell types that eventually leads to neuronal cell death and the manifestation of the AIDS-associated dementia complex (ADC). Upon autopsy HIV-infected individuals show lesions within subcortical regions of the brain, including the cerebellum. Previously we have demonstrated, in primary and cell culture models of rat and human astrocytes, a change in intracellular pH (pH(i)) due to increased Na(+)/H(+) exchange following exposure to inactivated virus or gp120, the major HIV envelope glycoprotein. To further investigate whether any such in vivo pH(i) changes occur in human brains subsequent to HIV infection, we measured the pH(i) of the cerebellum in eight HIV-positive individuals and nine healthy volunteers using (31)P magnetic resonance spectroscopy imaging (MRSI) at high field strength (4.1 T). The results showed a significant difference between the age-adjusted mean pH(i) in the cerebellum in control group and patient groups (7.11 +/- 0.03 vs 7.16 +/- 0.04), and further HIV-infected individuals displayed a significant increase in the number of cerebellar volume elements that were alkaline. We hypothesize that this propensity towards alterations in cerebellar pH(i) may portend later neurological involvement resulting from HIV infection.     

A role for Na+/H+ exchange in pH regulation in Helix neurones

We have used the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) to reexamine the mechanisms that extrude acid from voltage-clamped Helix aspersa neurones. Intracellular acid loads were imposed by three different methods: application of weak acid, depolarization and removal of extracellular sodium. In nominally CO2/HCO3-free Ringer the rate of recovery from acid loads was significantly slowed by the potent Na+/H+ exchange inhibitor 5-[N-ethyl-N-isopropyl]-amiloride (EIPA, 50 microM). Following depolarization-induced acidifications the rate of intracellular pH (pHi) recovery was significantly reduced from 0.41 +/- 0.13 pH units.h-1 in controls to 0.12 +/- 0.09 pH units.h-1 after treatment with EIPA at pHi approximately equal to 7.3 (n = 7). The amiloride analogue also reduced the rate of acid loading seen during extracellular sodium removal both in the presence and absence of the Na(+)-dependent Cl-/HCO3- exchange inhibitor 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid (SITS, 50 microM). This is consistent with EIPA inhibiting reverse-mode Na+/H+ exchange. In 2.5% CO2/20 mM HCO3-buffered Ringer pHi recovery was significantly inhibited by SITS, but unaffected by EIPA. Our results indicate that there are two separate Na(+)-dependent mechanisms involved in the maintenance of pHi in Helix neurones: Na(+)-dependent Cl-/HCO3- exchange and Na+/H+ exchange. Acid extrusion from Helix neurones is predominantly dependent upon the activity of Na(+)-dependent Cl-/HCO3- exchange with a lesser role for Na+/H+ exchange. This adds further weight to the belief that the Na+/H+ exchanger is ubiquitous.