Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

Rapid intracellular acidification and cell death by H2O2 and alloxan in pancreatic beta cells

Pancreatic beta-cell death induced by oxidative stress plays an important role in the pathogenesis of diabetes mellitus. We studied the relation between rapid intracellular acidification and cell death of pancreatic beta-cell line NIT-1 cells exposed to H2O2 or alloxan. Intracellular pH was measured by a pH-sensitive dye, and cell damage by double staining with Annexin-V and propidium iodide using flow cytometry. H2O2 and alloxan caused a rapid fall in intracellular pH and suppressed Na+/H+ exchanger activity in the NH4Cl prepulse method. H2O2 induced necrotic cell death, which shifted to apoptotic cell death when initial acidification was prevented by pH clamping to 7.4 using nigericin (unclamped cells vs clamped cells, necrosis 43.8 +/- 5.8% vs 21.1 +/- 10.6%, P < 0.05; apoptosis 8.0 +/- 1.9% vs 44.5 +/- 5.0%, P < 0.01). pH-clamped cells showed enhanced caspase 3 activity and proapoptotic Bax expression. On the other hand, NIT-1 cells were resistant to alloxan toxicity, but treatment with alloxan and nigericin strikingly enhanced the cytotoxicity. Antioxidants partly prevented cell death, although intracellular pH remained similarly acidic. The rapid intracellular acidification was not the cause of cell death but a significant determinant of the mode of death of H2O2 -treated beta cells, whereas no link between cell death and acidification was demonstrated in alloxan toxicity.     

The role of sodium ions in methanogenesis. Formaldehyde oxidation to CO2 and 2H2 in methanogenic bacteria is coupled with primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2

Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (delta G0' = -27 kJ/mol CO2), when methanogenesis was inhibited by 2-bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings. Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2-3 mol Na+/mol formaldehyde oxidized were extruded. Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores of Na+/H+ antiport inhibitors. Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores. Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+-translocating-ATP-synthase inhibitor, dicyclohexylcarbodiimide. With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.     

Mechanisms of pH regulation in the regulated secretory pathway

A precise pH gradient between organelles of the regulated secretory pathway is required for sorting and processing of prohormones. We studied pH regulation in live endocrine cells by targeting biotin-based pH indicators to cellular organelles expressing avidin-chimera proteins. In AtT-20 cells, we found that steady-state pH decreased from the endoplasmic reticulum (ER) (pH(ER) = 7.4 +/- 0.2, mean +/- S.D.) to Golgi (pH(G) = 6.2 +/- 0.4) to mature secretory granules (MSGs) (pH(MSG) = 5.5 +/- 0.4). Golgi and MSGs required active H(+) v-ATPases for acidification. ER, Golgi, and MSG steady-state pH values were also dependent upon the different H(+) leak rates across each membrane. However, neither steady-state pH(MSG) nor rates of passive H(+) leak were affected by Cl(-)-free solutions or valinomycin, indicating that MSG membrane potential was small and not a determinant of pH(MSG). Therefore, our data do not support earlier suggestions that organelle acidification is primarily regulated by Cl(-) conductances. Measurements of H(+) leak rates, buffer capacities, and estimates of surface areas and volumes of these organelles were applied to a mathematical model to determine the H(+) permeability (P(H+)) of each organelle membrane. We found that P(H+) decreased progressively from ER to Golgi to MSGs, and proper acidification of Golgi and MSGs required gradual decreases in P(H+) and successive increases in the active H(+) pump density.     

Proton translocation coupled to the oxidation of carbon monoxide to CO2 and H2 in Methanosarcina barkeri

Cell suspensions of acetate-grown Methanosarcina barkeri mediate the conversion of CO and H2O to CO2 and H2. The reaction is coupled with the phosphorylation of ADP. Evidence is presented that CO oxidation by the cells is associated with the transient acidification of the suspension medium. Up to 2 mol vectorial protons were measured/mol CO oxidized when the transmembrane electrical gradient was kept low by the addition of valinomycin (20 nmol/mg protein) and KCl (200 mM) or of KSCN (50 mM). No transient acidification was observed in the presence of the protonophore tetrachlorosalicylanilide which stimulated rather than inhibited CO oxidation. Proton extrusion remained unaltered when the proton-translocating ATPase was specifically inhibited by dicyclohexylcarbodiimide. The latter finding indicates that proton translocation is associated with CO conversion to CO2 and H2 rather than with ATP hydrolysis in the cells. The data substantiate that the coupling of CO oxidation with ADP phosphorylation in M. barkeri occurs via a chemiosmotic mechanism.     

Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++

Gap junctional conductance (gj) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with CO2 when extracellular calcium activity is near physiological levels (1.0 mM CaCl2 added; 470 microM Ca++). Intracellular calcium concentration (Cai), measured by fura-2 fluorescence in cell suspensions, was 148 +/- 39 nM (+/- SEM, n = 6) and intracellular pH (pHi), measured with intracellular ion-selective microelectrodes, was 7.05 +/- 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air. Cai increased to 515 +/- 12 nM (n = 6) and pHi decreased to 5.9-6.0 in medium equilibrated with 100% CO2. In air-equilibrated low-calcium medium (no added CaCl2; 2-5 microM Ca++), Cai was 61 +/- 9 nM (n = 13) at pHi 7.1. Cai increased to only 243 +/- 42 nM (n = 9) at pHi 6.0 in CO2-equilibrated low-calcium medium. Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pHi 5.9-6.0 in low-calcium medium. Cell pairs could still be electrically uncoupled reversibly by the addition of 100 microM octanol, an agent which does not significantly affect Cai. In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO2 increased Cai to 425 +/- 35 nM (n = 11) at pHi 5.9-6.0 and gj was reduced to near zero. Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187. The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification. In contrast, acidification did not substantially reduce gj when intracellular calcium was low. Increasing intracellular calcium did not appreciably reduce gj at pHi 7.0. These results suggest that, although other factors may play a role, H+ and Ca++ act synergistically to decrease gj.     

Activation of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae by addition of hydrogen peroxide.

Addition of hydrogen peroxide (greater than 10 mM) to aerated derepressed cells of S. cerevisiae in the absence of substrate caused a boost of endogenous respiration and both intra- and extracellular acidification, without any significant change in cellular ATP level. Furthermore, a hyperpolarization of the plasma membrane was indicated by an enhanced accumulation of tetraphenylphosphonium in the cells. The extracellular pH attained was as low as 3.5. The acidification could be suspended by the H(+)-ATPase inhibitors diethylstilbestrol and dicyclohexylcarbodiimide and was, in general, associated with an opposite flux of K+. K+ also stimulated the H(+)-ATPase activity in the purified plasma membrane fraction. These results are consistent with the plasma membrane H(+)-ATPase being involved in the H+ extrusion induced by H2O2 in the absence of substrate. Extended exposure of cells to H2O2 led eventually to an arrest of both respiration and ion fluxes that could be again lifted by depolarizing the plasma membrane. Along with differences in the cellular NADH/NAD+ ratio and in the participation of organic acids, this makes the H2O2-induced acidification distinct from that induced by glucose.     

Lobster hepatopancreatic epithelial single cell suspensions as models for electrogenic sodium-proton exchange

Sodium-proton antiporters, also called Na+/H+ exchangers (NHE), are vital transmembrane proteins involved in multiple cellular functions including transepithelial ion transport and Na+ homeostasis of cells throughout the biological kingdom. Na+/H+ exchange is accelerated by cytosolic acidification and also by osmotically induced cell shrinking, thereby promoting recovery of the physiological pHi and volume. Eight isoforms of Na+/H+ exchangers have been cloned and characterized to date and share the same overall structure, but exhibit differences with respect to cellular localization, kinetic variables and plasma membrane targeting, in polarized epithelial cells. The electrogenic Na+ absorption across tight epithelia from invertebrates follow significantly different principles from the electroneutral Na+/H+ antiporter found in vertebrates. In all invertebrate cells examined, the antiporter displayed a 2Na+/1H+ transport stoichiometry and this transport was markedly inhibited by exogenous calcium and zinc. Na+/H+ exchangers (NHE) are present in crustacean hepatopancreatic cell type suspensions and are believed to function in acid-base regulation by driving the extrusion of protons across the hepatopancreatic epithelium in exchange for Na+ in the sea water. A brief review of current knowledge about Na+/H+ exchangers has been presented. In addition, understanding of hepatopancreatic Na+/H+ exchange is described as obtained after isolation of purified E-, R-, F- and B-cell suspensions from the whole organ by centrifugal elutriation.     

Pulsed high-field gradient in vivo NMR spectroscopy to measure diffusional water permeability in Corynebacterium glutamicum

Pulsed high-field gradient in vivo NMR spectroscopy was used to measure diffusional water permeability in cell suspensions of the Gram-positive bacterium Corynebacterium glutamicum. Two different regions of H2O mobility were detected. One was characterized by the apparent coefficient of self-diffusion, D(1 app) = (4.6-12.7)x10(-8) cm(2) s(-1), depending on the observation time t. The other region was characterized by D(2) = 1.4x10(-5) cm(2) s(-1). The value of D(2) was similar to the diffusion coefficient of H2O in free water and in extracellular biological fluids. Restricted diffusion could be demonstrated for the slower process (D(1)). It was attributed to the cytoplasm of the cells. The membrane permeability, P(d H2O), for C. glutamicum was (4.8+/-0.4)x10(-3) cm s(-1). It compared favorably with values reported for human erythrocytes and was higher by a factor of about 100 compared to the diffusional permeability for ethanol, P(d ethanol), in Zymomonas mobilis. Addition of HgCl2, a water channel inhibitor in eukaryotes, decreased P(d H2O) in C. glutamicum by a factor of approximately 8. To our knowledge, these are the first functional studies of water transport in prokaryotes that yielded quantitative data, viz., transmembrane water permeability expressed through D(H2O) and P(d H2O).     

The dark respiration of Anacystis nidulans. Production of HCN from histidine and oxidation of basic amino acids.

The basic amino acids, L-arginine, L-lysine, LO-irnithine, and to a lesser extent L-histidine, strongly stimulate the O2 uptake of cell suspensions of the blue-green alga or cyanobacterium anacystis nidulans. In the case of L-histidine, the extra O2 consumption is associated with the formation in vivo of small amounts of HCN, particularly in an atmosphere of O2. The enzyme responsible for both the stimulated O2 uptake with the basic amino acids and the formation of HCN from histidine has been isolated and identified as an L-amino acid oxidase specific for the basic amino acids. The purification (15 000-fold) of this enzyme is described. The isolated enzyme is inhibited by o-phenanthroline, which has a similar inhibitory effect on the O2 uptake of cell suspensions with (and without) added amino acids. The basic amino acid oxidase, which is not inhibited by HCN, can be regarded as an 'alternate' oxidase in A. nidulans. An oxidase sensitive to HCN is apparently also operative. At high concentrations of lysine or arginine added HCN can almost double the initial rate of O2 consumption of cell suspensions. This can be attributed to the inhibition of catalase by HCN. At low concentrations of the amino acids, and with more prolonged incubation time, HCN becomes inhibitory. One interpretation could be that the HCN-sensitive terminal oxidase is also involved in the extra O2 uptake elicited by the basic amino acids, but other interpretations are possible. The extra O2 uptake elicited by histidine is almost completely inhibited by HCN, which is consistent with the finding that histidine is a relatively poor substrate for the basic amino acid oxidase.     

Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase

We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride (50 microM), indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+). Lowering pHb in the presence of oxytocin (50 mU/ml) produced a transepithelial current (3 microA.cm-2 at pHb 5.5) which was blocked by 100 microM of Hg2+, Zn2+, or Ni2+ at the basolateral side, and by DCCD (10(-5) M) or Hg2+ (100 microM) from the apical side. The net hydroosmotic water flux (JH2O) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H(+)-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10(-5) M), oligomycin (10(-8) M), and DCCD (10(-5) M) prevented JH2O when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10(-4) M), or 2-deoxyglucose (10(-3) M) had no effect on JH2O.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct measurement of the initial and early ratios of proton extrusion to oxygen uptake accompanying cytochrome c oxidation by rat liver mitoplasts.

We have recently described new methods that enable the sharp initiation of a respiratory pulse by photolysis of the CO complex of cytochrome oxidase in a stirred suspension of mitochondria, succinate, O2, and CO (Setty, O. H., R. I. Shrager, B. Bunow, B. Reynafarje, A. L. Lehninger, and R. W. Hendler. 1986. Biophys. J. 50:391-404). Data are collected directly into a microcomputer at 10-ms intervals from fast responding O2 and pH electrodes. These procedures eliminate delays and uncertainties due to mixing times, recorder response, and recovery of the O2 electrode from responding to the injection of O2. Correction procedures were also described for the inherent electrode delays. These procedures revealed an initial burst in medium acidification and a lag in O2 uptake that led to H+/O rates of 20-30 during the first 50 ms and relaxed to "normal" levels by 300 ms. Subsequent changes in [H+] and [O2] followed time courses that appeared to be, but were not strictly, first order. We describe here similar studies in which cytochrome c served as electron donor to site III of rat liver mitoplasts. A qualitatively similar but quantitatively smaller burst in medium acidification and H+/O ratio was seen in these studies. Implications of the previous (Setty et al., 1986) and current studies on defining "mechanistic" H+/O ratios are discussed.     

Cardiac performance of an isolated eel heart: effects of hypoxia and responses to coronary artery perfusion.

The pericardial sac containing the heart was removed from large (2.7-6.3 kg) long-finned eels (Anguilla dieffenbachii). Coronary arteries were cannulated in preparation for perfusion with eel Ringer or red cell suspensions. The hearts were maintained by Ringer perfusion while the performance of the heart was assessed. Responses of the hearts to increases in filling pressure and output pressure were recorded. Maximum cardiac output was 22.3 +/- 1.4 ml/min/kg body mass (mean +/- 1 SEM; N = 9). The highest cardiac power output was measured at maximum cardiac output and was 3.39 +/- 0.32 mW/g ventricle mass (mean +/- 1 SEM; N = 9). Eel hearts could sustain output pressures near 80 cm H2O, but cardiac output was reduced and cardiac power output was 1.89 +/- 0.24 mW/g ventricular mass (mean +/- 1 SEM; N = 9). Maximum cardiac output decreased by 14% when hearts pumped hypoxic Ringer with a PO2 of 11.5 torr. At high input pressures concomitant with high output pressures (80 cm H2O), cardiac power output decreased by 38% upon exposure to hypoxic Ringer. Coronary perfusion of hypoxic hearts with red cell suspensions (mean hematocrit 10.4%) at a rate of 2% of control cardiac output (0.20 ml/min/kg body mass) had no effect on maximum cardiac output. However, coronary perfusion enabled hypoxic hearts to maintain cardiac output when output pressure was raised to 80 cm H2O. Under conditions of high input pressure and high output pressure, power output increased by 20% compared to hypoxic hearts without coronary perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.     

Effect of renal medullary H2O2 on salt-induced hypertension and renal injury

Dahl salt-sensitive (SS) and consomic, salt-resistant SS-13(BN) rats possess substantial differences in blood pressure salt-sensitivity even with highly similar genetic backgrounds. The present study examined whether increased oxidative stress, particularly H2O2, in the renal medulla of SS rats contributes to these differences. Blood pressure was measured using femoral arterial catheters in three groups of rats: 1) 12-wk-old SS and consomic SS-13(BN) rats fed a 0.4% NaCl diet, 2) SS rats fed a 4% NaCl diet and chronically infused with saline or catalase (6.9 microg x kg(-1) x min(-1)) directly into the renal medulla, and 3) SS-13(BN) fed high salt (4%) and infused with saline or H2O2 (347 nmol x kg(-1) x min(-1)) into the renal medullary interstitium. After chronic blood pressure measurements, renal medullary interstitial H2O2 concentration ([H2O2]) was collected by microdialysis and analyzed with Amplex red. Blood pressure and [H2O2] were both significantly higher in SS (126 +/- 3 mmHg and 145 +/- 17 nM, respectively) vs. SS-13(BN) rats (116 +/- 2 mmHg and 56 +/- 14 nM) fed a 0.4% diet. Renal interstitial catalase infusion significantly decreased [H2O2] (96 +/- 41 vs. 297 +/- 52 nM) and attenuated the hypertension (146 +/- 2 mmHg catalase vs. 163 +/- 4 mmHg saline) in SS rats after 5 days of high salt (4%). H2O2 infused into the renal medulla of consomic SS-13(BN) fed high salt (4%) for 7 days accentuated the salt sensitivity (145 +/- 2 mmHg H2O2 vs. 134 +/- 1 mmHg saline). [H2O2] was also increased in the treated group (83 +/- 1 nM H2O2 vs. 44 +/- 9 nM saline). These data show that medullary production of H2O2 may contribute to salt-induced hypertension in SS rats and that chromosome 13 of the Brown Norway contains gene(s) that protect against renal medullary oxidant stress.     

The role of HCO3- and Na+/H+ exchange in the response of rat parotid acinar cells to muscarinic stimulation

The intracellular pH (pHi) of a rat parotid acinar preparation was monitored using the pH-sensitive fluorescent dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Under resting (unstimulated) conditions both Na+/H+ exchange and CO2/HCO3- buffering contribute to the regulation of pHi. Muscarinic stimulation (carbachol) of the acini produced a gradual rise in pHi (approximately 0.1 unit by 10 min) possibly due to activation of the Na+/H+ exchanger. When the exchanger was blocked by amiloride or sodium removal, carbachol induced a dramatic (atropine inhibitable) decrease in pHi (approximately 0.4 pH unit with t1/2 approximately 0.5 min at 1 mM carbachol). The rate of this acidification was reduced by removal of exogenous HCO3- and by the carbonic anhydrase inhibitor methazolamide. Also, acini stimulated with carbachol in Cl- -free solutions showed a more pronounced acidification than in the corresponding Cl- -replete media. Taken together, these data indicate that the carbachol-induced acidification of rat parotid acinar cells unmasked by inhibition of the Na+/H+ exchanger is due to a rapid loss of intracellular HCO3-. Carbachol induced acidification was inhibited by the Cl- channel blocker diphenylamine 2-carboxylate but not by 4-acetomido-4'-isothiocyanostilbene-2,2'-disulfonic acid, an inhibitor of Cl-/HCO3- exchange. In addition, this acidification could not be sustained in Ca2+-free media and was totally blocked by chelation of intracellular Ca2+. Interpreted in terms of HCO3- loss, these results closely parallel the pattern of carbachol-induced Cl- release from this same preparation and indicate that HCO3- is secreted in response to muscarinic stimulation via the same or a very similar exit pathway, presumably an apical anion channel. Under normal physiological conditions the intracellular acidification resulting from HCO3- secretion is buffered by the Na+/H+ exchanger.     

Oxidative phosphorylation and energy buffering in cyanobacteria.

The onset of respiration in the cyanobacteria Anacystis nidulans and Nostoc sp. strain Mac upon a shift from dark anaerobic to aerobic conditions was accompanied by rapid energization of the adenylate pool (owing to the combined action of ATP synthase and adenylate kinase) and also the guanylate, uridylate, and cytidylate pools (owing to nucleoside diphosphate and nucleoside monophosphate kinases). Rates of the various transphosphorylation reactions were comparable to the rate of oxidative phosphorylation, thus explaining, in part, low approximately P/O ratios which incorporate adenylates only. The increase of ATP, GTP, UTP, and CTP levels (nanomoles per minute per milligram [dry weight]) in oxygen-pulsed cells of A. nidulans and Nostoc species was calculated to be, on average, 2.3, 1.05, 0.8, and 0.57, respectively. Together with aerobic steady-state pool sizes of 1.35, 0.57, 0.5, and 0.4 nmol/mg (dry weight) for these nucleotides, a fairly uniform turnover of 1.3 to 1.5 min-1 was derived. All types of nucleotides, therefore, may be conceived of as being in equilibrium with each other, reflecting the energetic homeostasis or energy buffering of the (respiring) cyanobacterial cell. For the calculation of net efficiencies of oxidative phosphorylation in terms of approximately P/O ratios, this energy buffering was taken into account. Moreover, in A. nidulans an additional 30% of the energy initially conserved in ATP by oxidative phosphorylation was immediately used up by a plasma membrane-bound reversible H+-ATPase for H+ extrusion. Consequently, by allowing for energy buffering and ATPase-linked H+ extrusion, maximum P/O ratios of 2.6 to 3.3 were calculated. By contrast, in Nostoc sp. all the H+ extrusion, appeared to be linked to a plasma membrane-bound respiratory chain, thus bypassing any ATP formation and leading to P/O ratios of only 1.3 to 1.5 despite the correction for energy buffering.     

Quantifying rates of protein synthesis in humans by use of 2H2O: application to patients with end-stage renal disease

A method is introduced for quantitating protein synthetic rates in humans by use of (2)H(2)O. Its validity was tested in subjects with end-stage renal disease. Six clinically stable subjects, hemodialyzed three times weekly, ingested (2)H(2)O to a body water (2)H enrichment of approximately 0.4%. On dialysis, body water enrichment declined to approximately 0.1%. Enrichment of the alpha-hydrogen of plasma free alanine was also approximately 0.4% before and approximately 0.1% after dialysis. Beta-hydrogen enrichment was approximately 80-100% of alpha-hydrogen enrichment. (2)H(2)O was ingested to replace (2)H(2)O removed after each dialysis for 15-51 days, returning enrichment to approximately 0.4%. Enrichment of alanine from plasma albumin gradually increased, with again approximately 80-100% as much (2)H in beta- as in alpha-hydrogens. With continued dialyses, without (2)H(2)O replacement, alanine from albumin enrichment gradually declined, whereas free alanine and water enrichments were negligible. The fractional albumin synthesis rate, calculated from the increase in enrichment in alanine from albumin, was 4.0 +/- 0.5%/day, and from the decrease, 4.6 +/- 0.2%/day. Thus body water enrichment in a subject given (2)H(2)O can be maintained constant long term. A rapid exchange, essentially complete, occurs between the hydrogens of alanine and body water. An integrated measure over a long period of albumin's synthetic rate can be estimated from both the rise in enrichment of alanine from the protein during (2)H(2)O ingestion and fall on (2)H(2)O withdrawal, while the subject's living routine is uninterrupted. Estimates are in subjects with renal disease, but the method should be applicable to estimates of protein synthetic rates in normal subjects and in other pathological states.     

Phosphorus-31 nuclear magnetic resonance studies of bioenergetics in wild-type and adenosinetriphosphatase(1-) Escherichia coli cells

By use of 31P NMR, the transmembrane pH gradient (delta pH) and the intracellular levels of phosphorylated metabolites were measured in aerobic suspensions of wild-type Escherichia coli cells in the presence and absence of the adenosinetriphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide (DCCD); the same parameters were also determined in E. coli mutants deficient in ATPase activity under both anaerobic and aerobic conditions. A method is described by which dense suspensions of E. coli cells (approximately 3 X 10(11) cells/mL) were oxygenated so that steady-state O2 levels in the suspensions were far greater than the Km for O2 consumption. Under these conditions, in wild-type MRE600 cells, the intracellular concentrations of PI, NTP, and NDP were measured to be 3.0 +/- 1.5, 8 +/- 1, and 1.2 +/- 1 mM, respectively, while the intracellular pH was approximately 7.5 over the external pH range studied (6 to approximately 7.0). Upon treatment with DCCD, the intracellular NTP level was drastically reduced and intracellular Pi concentration increased in respiring wild-type cells; in the same cells, however, DCCD did not affect the intracellular pH and the delta pH. During respiration in the presence of lactate, ATPase- cells established a delta pH but failed to synthesize any detectable levels of NTP. Conversely, ATPase- cells accumulated high levels of NTP but did not generate a delta pH during glycolysis under anaerobic conditions. These results are in complete agreement with the generally accepted chemiosmotic hypothesis. 31P NMR data on intact ATPase- NR70 cells were in agreement with the previously proposed [Rosen, B. P., Brey, R., & Hasan, S. (1978) J. Bacteriol. 134, 1030] existence of a proton leak in this strain which is sealed by DCCD or by spontaneous mutation into strain NR71. However, the NMR data also indicated that other major differences exist between NR71 and NR70 cells.