Anion channels transport ATP into the Golgi lumen

Anion channels provide a pathway for Cl^– influx into the lumen of the Golgi cisternae. This influx permits luminal acidification by the organelle's H⁺-ATPase. Three different experimental approaches, electrophysiological, biochemical, and proteomic, demonstrated that two Golgi anion channels, GOLAC-1 and GOLAC-2, also mediate ATP anion transport into the Golgi lumen. First, GOLAC-1 and -2 were incorporated into planar lipid bilayers, and single-channel recordings were obtained. Low ionic activities of K₂ATP added to the cis-chamber directly inhibited the Cl^– subconductance levels of both channels, with K_m values ranging from 16 to 115 µM. Substitution of either K₂ATP or MgATP for Cl^– on the cis, trans, or both sides indicated that ATP is conducted by the channels with a relative permeability sequence of Cl^– > ATP^4– > MgATP^2–. Single-channel currents were observed at physiological concentrations of Cl^– and ATP, providing evidence for their importance in vivo. Second, transport of [-³²P]ATP into sealed Golgi vesicles that maintain in situ orientation was consistent with movement through the GOLACs because it exhibited little temperature dependence and was saturated with an apparent K_m = 25 µM. Finally, after transport of [-³²P]ATP, a protease-protection assay demonstrated that proteins are phosphorylated within the Golgi lumen, and after SDS-PAGE, the proteins in the phosphorylated bands were identified by mass spectrometry. GOLAC conductances, [-³²P]ATP transport, and protein phosphorylation have identical pharmacological profiles. We conclude that the GOLACs play dual roles in the Golgi complex, providing pathways for Cl^– and ATP influx into the Golgi lumen. Golgi complex; Cl channel; mass spectrometry; phosphorylation     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

The Ionic Relations of Acetabularia mediterranea

The concentrations of K⁺, Na⁺, and Cl^– in the cytoplasmand the vacuole of Acetabularia mediterranea have been measured,as have the vacuolar concentrations of SO₄^–– andoxalate. The electrical potential difference between externalsolution, and vacuole and cytoplasm has been measured. The resultsindicate that Cl^– and SO₄^–– are probably transportedactively into the cell, and that active transport of Na⁺ isoutwards. The results for K⁺ are equivocal. The fluxes of K⁺,Na⁺, Cl^–, and S0₄^–– into the cell and theeffluxes of Na⁺ and Cl^– have been determined. The Cl^–fluxes are extremely large. In all cases the plasmalemma isthe rate-limiting membrane for ion movement. A technique isdescribed for the preparation of large, completely viable cellfragments containing only cytoplasm, with no vacuole.     

Ionic Currents across the Plasmalemma of Chara inflata Cells: I. OSMOTIC EFFECTS OF SORBITOL ON K+, CI- AND LEAK CURRENTS

This paper describes experiments designed to investigate theeffects of increases in external osmotic pressure on the electrophysiologicalbehaviour of the plasmalemma in cells of the brackish-wateralga, Chara inflata. The electrical conductance of the plasmalemmaof these cells of, due to the diffusion of ions, consists mainlyof K⁺, Cl^– and leak components. The addition of sorbitolat concentrations in the range 40–280 mol m^–3 tothe external solution bathing the cells, progressively and reversiblydepolarized the cell membrane and increased the total membraneconductance, chiefly due to increases in each of the separateionic conductances. At concentrations higher than about 280mol m^–3 when the cells became plasmolysed, the effectsof sorbitol on the electrical properties of the cell ceasedto be fully reversible. When the membrane electrical potentialdifference is stepped in a negative direction with a voltage-clamp,the resulting inward current has voltage-dependent componentsconsisting of an inactivating K⁺ current, an activating Cl^–current and a constant leak current. The addition of sorbitolto the external solution modified these currents by increasingtheir magnitude, by increasing the half-time of the inactivationof the K⁺ current, and by decreasing the half-time of activationof the Cl^– current. Key words: Chara inflata, osmotic effects, K⁺ and Cl^– currents     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.     

Anion-dependent changes of energy transfer in chloroplasts III. Evidence for inactivation of light-triggered adenosine triphosphatase in chloroplasts by chloride

Washing chloroplasts with a high concentration of Tris-Cl^- buffercaused Cl^- dependent inhibition of photophosphorylation, light-inducedpH rise and light-triggered Mg²⁺-dependent ATPase activity.The inhibition of these activities was largely prevented bythe presence of 10^–4 M ADP or ATP during Tris washing,especially that of Mg²⁺-ATPase activity. The results were interpretedas suggesting that the inactivation of light-triggered ATPaseactivity in chloroplasts by chloride is one of the causes ofthe uncoupling of chloroplasts with Tris washing. (Received April 30, 1974; )     

Phosphate Relations of Acetabularia: Phosphate Pools, Adenylate Phosphates and 32P Influx Kinetics

Steady state phosphate relations are determined for the marinealga Acetabularia mediterranea with respect to cellular phosphatepools and phosphate transport. About 20% of the cellular acid-labilephosphate is found in the cell wall fraction (low speed sediment).By the use of cytoplasm-depleted cell segments, it isa establishedthat only about 10% of the total intracellular phosphate islocalized in the vacuole when cells are bathed in normal phosphateconcentration (<30 µM). Measurements of ATP, ADP, AMPand inorganic phosphate (Pj) in the entire cytoplasm and inisolated chloroplasts has enabled the calculation of the cytosolicadenylate energy charge (0.7–0.9) and phosphate potential(110–170 mV). Influx of ³²P, displays complex kinetics.Four components for uptake (approximate time constants: (A)1 s, (B) 20 s, (C) 300 s and (D) 3000 s) are tentatively identified.Focusing on the faster components, B and C, and possibly evenA, appear to be metabolically-linked, as judged by their sensitivityto temperature and to the inhibitor 2, 4-dinitrophenol. Thesethree components of influx are proportional to the externalP, concentration for values <30 µM, but B and C tendto saturate at higher concentrations. The results are discussedwith respect to the energetics of transport at the plasmalemmaof Acetabularia, especially the activity of the electrogenicCI-ATPase. Key words: Acetabularia, Energy charge, Phosphate pools, Phosphate potential, Phosphate uptake kinetics     

Electrogenic Pump Current and ATP-Dependent H+ Efflux Across the Plasma Membrane of Nitellopsis obtusa

The correlation between the pump current and the ATP-dependentH⁺ efflux was examined in internodal cells of Nitellopsis obtusa.To control the cytoplasmic pH and ATP concentration, the tonoplastwas removed by intracellular perfusion with an EGTA-containingmedium. Two groups of perfused cells were prepared, one with1 mM ATP (+ATP cells) and the other without ATP but with hexokinaseand glucose (–ATP cells). The ATP-dependent H⁺ effluxwas calculated as the difference in H⁺ efflux between the +ATPand –ATP cells. Based on an electrically equivalent circuitmodel of the plasma membrane, the pump current was calculatedfrom the membrane potentials and the membrane resistances ofboth +ATP and –ATP cells. When the membrane potentialwas not too high (–220 mV), the ATP-dependent H⁺ current(19 mA m^–2) was almost equal to the pump current (20 mAm^–2) calculated from the electrical data. This indicatesthat the electrogenic pump current across the plasma membraneof Nitellopsis obtuse was mostly carried by H⁺. But when themembrane potential was high (–280 mV), the H⁺ currentwas lower than the pump current. The possible cause of thisdiscrepancy is discussed. (Received November 5, 1984; Accepted February 28, 1985)     

Cl- Fluxes and Cl- Content of Dunaliella acidophila--An Alga with a Positive Membrane Potential

The Cl^– fluxes across the plasma membrane and the Cl^–content of the acid–resistant alga Dunaliella acidophila(optimal growthat pH 1.0, positive membrane potential) werestudied in the presence of 0.01–300 mM Cl^–. Up to40 mM Cl^– in the medium, theinternal Cl^– concentrationis higher than that predicted by the electrochemical equilibrium,whereas at higher external Cl^– concentrations internalCl^– levels are lower than expected for the electrochemicalequilibrium. Growth in the absence of Cl^– is significantlylower than in the standard growth medium (2.2 mM Cl^–)and this reduction cannot be overcome by the addition ofothermonovalent anions such as Br^– or NO^–₃ The latterimplies a specific Cl^– requirement in addition to therole of Cl^– as apermeant anion during ion translocations.Growth and photosynthesis tolerate an excess of Cl^– upto 300 mM (without stepwiseadaptation to increasing salinity).The uptake of Cl^– (measured by tracer techniques) exhibitsMichaelis–Menten kinetics (K_M = 0.75 mM Cl^–) andis stimulated by light and high H⁺ concentrations. Internalacidification by acetic acid causes an inhibition of Cl^–uptake. The uptake of Cl^– is inhibited by the monovalentanions Br^–, I^–, and NO^–₃ with K¹, values notvery much different from the K_M. value for Cl^–. The aniontransport inhibitors SITS and DIDS do not affect photosynthesis,but strongly suppressthe uptake of Cl^–. The Cl^–channel blockers A–9–C and NPPB cause inhibitionsof Cl^– uptake as well as of photosynthesis andthe ATPpool. FCCP strongly depresses the internal ATP–pool withouta marked effect on Cl^– uptake. Cl^– efflux was inhibitedbyDIDS and SITS, but stimulated or inhibited by FCCP, dependingon the external Cl^– concentration. Results are in agreementwiththe hypothesis that Cl^– uptake into D. acidophila is dueto catalysed diffusion and is primarily independent of the hydrolysisofATP. Cl^– efflux is assumed to be coupled to an activepump. Data suggest tight co–operativity between the systemsresponsiblefor Cl^– uptake and Cl^– efflux, with thecytoplasmic pH and the membrane potential being important mediators. Key words: Acid resistance, chloride carrier, chloride channels, Dunaliella acidophila, membrane potential, plasma membrane     

Electrophysiological Evidence for an Electrogenic Proton Pump and the Proton Symport of Glucose in the Marine Fungus Dendryphiella salina

The marine hyphomycete Dendryphiella salina (Suth.) Nicot &Pugh has a resting membrane potential of –250 mV (insidenegative). The respiratory inhibitors sodium azide and FCCPinduced a rapid but reversible depolarization of the membraneof at least 180 mV; sodium azide also caused alkalinizationof the medium. Vanadate brought about significant depolarizationbut this was not always reversible. EDTA induced depolarizationthough to a lesser extent. DIDS and SITS caused a depolarizationof around 30–70 mV which was readily reversible, N-ethylmaleimideirreversibly depolarized the membrane by 180–200 mV. Ouabainhad no effect. When external concentrations of H⁺ , K⁺ , Na⁺or Cl^– were changed singly, only changes in H⁺ affectedmembrane potential, with shifts decreasing with increasing pH.Glucose and 3-O-methyl glucose depolarized the membrane in aconcentration-dependent manner which was enhanced by starvationof the hyphae. Recovery occurred in the presence of the hexose.Glucose caused an alkalinization of the medium, with time characteristicssimilar to the membrane potential changes. It is concluded thatthere is an electrogenic proton pump and a proton—glucosesymporter in D. salina. The retention of proton-based transportsystems suggests a terrestrial origin for the fungus. Key words: Marine fungi, Dendryphiella salina, membrane potential, electrogenic proton pump, proton symport, hexose     

Effect of Abscisic Acid on Sorbitol Uptake in Growing Apple Fruits

Levels of abscisic acid (ABA) in the fruit flesh of developingapples (cv. Golden Delicious) were measured by electron capturegas chromatography. ABA content of the tissue, calculated ona fresh weight basis, decreased at a constant rate from 200µg g^–1 in young fruit to 10 µg g^–1 inolder fruit and then increased when the ripening process commenced.On a whole fruit basis, the ABA level increased during the initialphase of fruit growth, remained constant during the linear growthphase and increased again when fruits started to ripen. During fruit development the ABA content correlated with therate of sorbitol uptake, when measured after discs of fruittissue were incubated in [¹⁴C]sorbitol. Sorbitol uptake washigh during the initial growth phase and declined at a constantrate during fruit development. ABA present in the incubation medium stimulated sorbitol uptakeinto fruit tissue at concentrations higher than 10^–8 M,whereas indolyl-3-acetic acid had no effect on uptake. When comparing sorbitol uptake in different zones of young fruit,it was found that uptake was higher in discs of outer fruitlayers than in discs from inner fruit zones. Key words: Pyrus malus, Apple, Abscisic acid, Sorbitol     

Chloride Transport in Chara: I. KINETICS AND CURRENT-VOLTAGE CURVES FOR A PROBABLE PROTON SYMPORT

Chara cells show an inward positive electric current acrossthe plasmalemma when exposed to Cl^– under voltage-clampconditions. The rapid rise of this current suggests that itis directly associated with the inward transport of Cl^–.The dependence of the current on Cl^– concentration showssaturation, the data fitting the Michaelis-Menten equation withV_m up to 100 nmol m^–2 s^–1 (for Cl^–starvedcells) with K_M 10–20 µM, and with some allowancefor an unstirred layer of water adjacent to the membrane. Theeffects on the current of clamp potential, illumination, withdrawalof alkali metal cations, and addition of amine were also investigated.These results suggest that the mechanism is the symport of 2H⁺ with each Cl^–, and that the actions of light, externalK⁺, and amine in stimulating Cl^–, influx are indirect.     

Measurements of Ion Concentrations inDunaliella parvaSubjected to Hypertonic Shock

Cells of Dunaliella parva, a green halotolerant alga, were equilibratedin 0.5 M NaCl (the same concentration as in the growth medium).The time – course of changes in K⁺, Na⁺, Cl^–, andpellet volume were followed after a hypertonic shock causedby increasing the outside NaCl concentration to 1 M. The cellsresponded initially by shrinking; they returned to their originalvolume in the course of the next 2–3 h. At the time ofthe hypertonic shock there were rapid influxes of Na⁺ and Cl^–which took about 30 min for completion. Thereafter, there wereprogressive losses of Na⁺ and Cl^–; these losses were temperature-dependentand presumably activated by metabolic energy. Cell K⁺ remainedconstant throughout.     

Fluxes of Sodium and Potassium in Acetabularia mediterranea

Sodium efflux in Acetabularia mediterranea occurs against agradient of electrochemical potential and is a light-stimulated,temperature-sensitive process; it is not sensitive to the uncouplerCCCP. Sodium influx is stimulated in CCCP and at low temperature.Potassium influx is temperature- and uncoupler-sensitive, butis not light-stimulated. Tracer K efflux shows complex kinetics,which cannot be explained by any arrangement of intracellularcompartments; it appears to be stimulated at low temperatureand is insensitive to light and uncouplers. There is no evidencefor any chemical linkage between fluxes of Na⁺, K⁺, or Cl^–.It is concluded that Na^– efflux at the plasmalemma isan active process, but no consistent explanation can be advancedto account for the results of K⁺ flux measurements.     

Effects of Ammonia and Methylamine on Cl- Transport and on the pH Changes and Circulating Electric Currents Associated with HCO3- Assimilation in Chara corallina

Low concentrations of ammonia and methylamine greatly increaseCl^– influx into Chara corallina. Both amines have theirmaximum effect at pH 6.5–7.5. The amine stimulation ofCl^– influx is small below about pH 5.5. Above pH 8.5 theremay be inhibition of influx by amines. Concentrations of 10–25µM ammonia are sufficient to cause the maximum stimulationof Cl^– influx; the corresponding methylamine concentrationsare 0.1–0.2 mM. It is concluded that entry of amine cations(NH₄^$ and CH₃NH₃^$), rather than unionized bases (NH₃ and CH₃NH₂),causes Cl^– transport to be increased. Increases in rates of Cl^– transport are not necessarilyaccompanied by effects on HCO₃^$ assimilation and OH^– efflux.Measurements of localized pH differences at the cell surfaceand of circulating electric currents in the bathing solutionshow that these phenomena are only significantly affected byammonia at or above 50 µM and by methylamine at or above1.0 mM. The significance of the effects of amines is assessedin relation to current ideas about transport of Cl^–, HCO₃^–,and OH^–.     

Light-induced changes in membrane potential in Spirogyra

Spirogyra cells exhibited changes in membrane potential whenthey were exposed to light. Cells made chloroplast-free didnot show any light-induced potential change (LPC) upon illuminationwith white light and also monochromatic red (680 nm) and farred (720 nm) light. LPC was observed when the cell containedonly a small fragment of chloroplast, whether the cell had anucleus or not. The magnitude of LPC depended on the amountof chloroplast in the cell. DCMU at 10^–5 M, CCCP at 10^–5 M and DNP at 10^–4M at pH 5.5 suppressed LPC, while CCCP at 1–5 ? 10^–6M, NH₄Cl at 5 ? 10^–2 M and DNP at 10^–4 M at pH 7.0stimulated LPC. PMS at 10^–4 M stimulated LPC and couldinduce LPC which was completely inhibited by DCMU. These factssuggest that LPC is related to noncyclic and cyclic electronflows. The influences of light and dark conditions and various metabolicinhibitors (DCMU, DNP, CCCP, NH₄Cl) on ATP level have been investigated.No significant difference in the ATP level was observed betweencells in the light and dark. DNP at 10^–4 M (pH 5.5) andCCCP at 5 ? 10^–6 M decreased the ATP level significantly,while DCMU and NH₄Cl only slightly. Good correlation was notfound between the total ATP level and LPC in Spirogyra. LPC occurred even when the external medium contained only asingle salt such as KCl, NaCl or CaSO₄. LPC was also recorded in chloroplasts in situ and in vitro.The mode of LPC of chloroplasts was quite different from thatof the cell. On illumination, the chloroplast potential changedvery rapidly and transiently in the positive direction thenrecovered spontaneously to almost the original potential level. Possible causes of LPC are discussed in relation to the electrogenicion pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan. (Received November 9, 1977; )     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

Nitrate Inhibition of Chloride Influx in Barley: Implications for a Proposed Chloride Homeostat

Net accumulation of Cl^– by intact barley plants was virtuallyeliminated in roots and reduced by 40% in shoots when externalmedia (0.5 mol m^–3 CaSO₄ plus 0–5 mol m^–3KCI) were supplemented with 0.25 mol m^– Ca(NO₃)₂. Plasmalemma³⁶Cl^– influx (_oc) was shown to be insensitive to externalNO₃^- in plants which had previously been grown in solutionslacking ^–3, but _oc became sensitive to NO₃^-after a lagperiod of 3–6 h. Kinetic analyses revealed that the inhibitionof 36C1^– influx by external NO₃^- was complex. At 0.25mol m^–3 NO₃^- the V_max for Cl^– influx was reducedby greater than 50%, with insignificant effects upon K_m. At0.5 mol m^–3 NO₃^- there was no further effect upon V_maxbut K_m for influx increased from 38±5 mmol m^–3to 116±26 mmol m^–3. By contrast, Cl^– effluxwas found to be insensitive to external NO₃^-. A model for theregulation of Cl^– influx is proposed which involves bothnegative feedback effects from vacuolar NO₃^- +Cl^–) concentrationand (external) NO₃^- inhibition of Cl^– influx at the plasmalemma.These combined effects serve to discriminate against Cl^–accumulation, favouring NO₃^- accumulation, when the latter ionis available. Such observations are inconsistent with recentproposals for the existence of bona fide homeostats for chlorideaccumulation in higher plants. Key words: Nitrate inhibition, Chloride influx, Barley     

Relocalization of the V-ATPase B2 subunit to the apical membrane of epididymal clear cells of mice deficient in the B1 subunit

An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H⁺ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1^–/– mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1^–/– mice compared with B1^+/+. Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1^–/– mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1^–/– mice as in B1^+/+ (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1^–/– mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1^–/– mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility. male reproductive tract; male fertility; luminal acidification; proton pump; vacuolar H⁺ATPase     

Three distinct mechanisms of HCO3- secretion in rat distal colon

HCO₃^– secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO₃^– secretion have revealed evidence of lumen Cl^– dependence, suggesting a role for apical membrane Cl^–/HCO₃^– exchange, direct examination of HCO₃^– secretion in isolated crypt from rat distal colon did not identify Cl^–-dependent HCO₃^– secretion but did reveal cAMP-induced, Cl^–-independent HCO₃^– secretion. Studies were therefore initiated to determine the characteristics of HCO₃^– secretion in isolated colonic mucosa to identify HCO₃^– secretion in both surface and crypt cells. HCO₃^– secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO₃^– secretion (5.6 ± 0.03 µeq·h^–1·cm^–2) was abolished by removal of either lumen Cl^– or bath HCO₃^–; this Cl^–-dependent HCO₃^– secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h^–1·cm^–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl^– channel blocker. 8-Bromo-cAMP induced Cl^–-independent HCO₃^– secretion (and also inhibited Cl^–-dependent HCO₃^– secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl^–-independent, DIDS-insensitive, saturable HCO₃^– secretion that was not inhibited by NPPB. Three distinct HCO₃^– secretory mechanisms were identified: 1) Cl^–-dependent secretion associated with apical membrane Cl^–/HCO₃^– exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO₃^– exchange. chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat