Effects of pCai and pHi on cell-to-cell coupling

Summary Internal longitudinal resistance (r_i), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H⁺ and Ca²⁺ may modulate r_i is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure r_i during various interventions. In some experiments, intracellular pH (pH_i) was recorded simultaneously to study the pH_i-r_i relation. Both intracellular Ca²⁺ and H⁺ independently modified r_i. However, internal resistance of cardiac fibers was insensitive to pH_i changes compared to other tissues. A latent period preceded the pH_i-related changes in r_i and the amount of change depended upon methodology. The results suggest that direct action of protons on r_i may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H⁺- or Ca²⁺-dependent phosphorylation of junctional proteins.     

Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family

Bacterial Trk and Ktr, fungal Trk and plant HKT form a family of membrane transporters permeable to K⁺ and/or Na⁺ and characterized by a common structure probably derived from an ancestral K⁺ channel subunit. This transporter family, specific of non-animal cells, displays a large diversity in terms of ionic permeability, affinity and energetic coupling (H⁺–K⁺ or Na⁺–K⁺ symport, K⁺ or Na⁺ uniport), which might reflect a high need for adaptation in organisms living in fluctuating or dilute environments. Trk/Ktr/HKT transporters are involved in diverse functions, from K⁺ or Na⁺ uptake to membrane potential control, adaptation to osmotic or salt stress, or Na⁺ recirculation from shoots to roots in plants. Structural analyses of bacterial Ktr point to multimeric structures physically interacting with regulatory subunits. Elucidation of Trk/Ktr/HKT protein structures along with characterization of mutated transporters could highlight functional and evolutionary relationships between ion channels and transporters displaying channel-like features.     

The regulation of ion channels and transporters by glycolytically derived ATP

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na⁺/K⁺-ATPase, the H⁺-ATPase, various types of Ca²⁺-ATPases, the Na⁺/H⁺ exchanger, the ATP-sensitive K⁺ channel, cation channels, Na⁺ channels, Ca²⁺ channels and other channels involved in intracellular Ca²⁺ homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches. Received 20 July 2007; received after revision 30 July 2007; accepted 17 August 2007     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Alkalosis and renal excretion of ammonia by rat kidney

Summary Upon sulfate administration, U_pH falls more in alkalotic rats than in controls. Alkalosis can lead to a reduction in U_{NH 3}V at highly acidic urine. The significance of this process is doubtful at U_pH ranging from about 6 to 7. At lower U_pH less NH₃ would be excreted, thereby less H⁺ would be trapped in urine and some acid would be conserved.     

Biochemical events associated with rapid cellular damage during the oxygen-and calcium-paradoxes of the mammalian heart

Summary The O_2– and Ca²⁺-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H⁺ and e^– and also oxygen radicals or recox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca²⁺ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised condition, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca²⁺ or by raising [Ca²⁺]_i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca²⁺-paradox by the membrane perturbation associated with removal of extracellular Ca²⁺; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca²⁺]_i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca²⁺-paradox which continues under anoxia. Massive entry of Ca²⁺ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Regulation of the H<Superscript>+</Superscript>-ATP synthase by IF1: a role in mitohormesis

The mitochondrial H⁺-ATP synthase is a primary hub of cellular homeostasis by providing the energy required to sustain cellular activity and regulating the production of signaling molecules that reprogram nuclear activity needed for adaption to changing cues. Herein, we summarize findings regarding the regulation of the activity of the H⁺-ATP synthase by its physiological inhibitor, the ATPase inhibitory factor 1 (IF1) and their functional role in cellular homeostasis. First, we outline the structure and the main molecular mechanisms that regulate the activity of the enzyme. Next, we describe the molecular biology of IF1 and summarize the regulation of IF1 expression and activity as an inhibitor of the H⁺-ATP synthase emphasizing the role of IF1 as a main driver of energy rewiring and cellular signaling in cancer. Findings in transgenic mice in vivo indicate that the overexpression of IF1 is sufficient to reprogram energy metabolism to an enhanced glycolysis and activate reactive oxygen species (ROS)-dependent signaling pathways that promote cell survival. These findings are placed in the context of mitohormesis, a program in which a mild mitochondrial stress triggers adaptive cytoprotective mechanisms that improve lifespan. In this regard, we emphasize the role played by the H⁺-ATP synthase in modulating signaling pathways that activate the mitohormetic response, namely ATP, ROS and target of rapamycin (TOR). Overall, we aim to highlight the relevant role of the H⁺-ATP synthase and of IF1 in cellular physiology and the need of additional studies to decipher their contributions to aging and age-related diseases.     

The role of calcium in aggregation and development ofDictyostelium

Changes in cytosolic Ca²⁺ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca²⁺]_i) at a low level in the face of steep gradients of extracellular and sequestered Ca²⁺. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca²⁺ into the cytosol and elevation of [Ca²⁺]_i. After such stimulation Ca²⁺ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca²⁺]_i to its basal level. Such responses to elevation of [Ca²⁺]_i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.     

Increased Na+-H+ exchanger activity in the ileal brush-border membrane of spontaneously hypertensive rats

In the present study, we have examined the intestinal Na⁺ transport, through the Na⁺-H⁺ exchanger, in ileal brush-border membrane vesicles (BBMV) isolated from spontaneously hypertensive rats (SHR), and normotensive Wistar Kyoto (WKY) rats as a control group. Na⁺ uptake into ileal BBMV was stimulated in the presence of a proton gradient (pH 5.5 inside/pH 7.5 outside) in SHR and WKY rats, resulting in a transient accumulation (overshoot) in both groups of rats. No overshoot was observed in the absence of a pH gradient. The magnitude of the accumulation was significantly higher in SHR than in WKY rats. Uptake of Na⁺ at equilibrium was identical in the presence and the absence of a proton gradient and was not changed in SHR. The use of amiloride inhibited pH gradient-driven Na⁺ uptake in a dose-dependent manner with a Ki of 90 μM and 100 μM for SHR and WKY rats, respectively. The relationship between proton gradient-driven Na⁺ uptake and external Na⁺ concentration was saturable and conformed to Michaelis-Menten kinetics in both SHR and WKY rats. Lineweaver-Burk analysis of the pH gradient-driven Na⁺ uptake indicated values of V_max that were significantly increased in SHR compared to WKY rats (11.4±0.55 nmol/mg/8 s vs. 4.96±0.78 nmol/mg/8 s for SHR and WKY rats, respectively). In contrast, similar K_m values for Na⁺ were found between SHR and WKY rats (4.0±0.2 mM vs. 4.9±0.6 mM for SHR and WKY rats, respectively). These studies show derangement in ileal BBMV Na⁺ transport of SHR, which is characterized by increased Na⁺-H⁺ exchanger activity. Received 18 December 1996; received after revision 3 February 1997; accepted 7 February 1997     

Stimulation of H+ ion secretion from the isolated mouse stomach by sodium fluoride

Summary The effect of sodium fluoride on H⁺ ion secretion was investigated in the isolated distended mouse stomach. It was found that sodium fluoride on its own caused dose-related stimulation of H⁺ ion secretion. Sodium fluoride did not inhibit H⁺ ion secretion induced by histamine. The possible mechanisms involved are discussed. It is considered that sodium fluoride might stimulate H⁺ ion secretion by causing histamine release and by increasing cyclic AMP formation in the intact gastric mucosa.     

Cell-to-cell coupling studied in isolated ventricular cell pairs

Summary Cell pairs isolated from adult rat and guinea pig ventricles were used to study the electrical properties of the nexal membrane. Each cell of a pair was connected to a voltage-clamp system so as to enable whole-cell, tight-seal recording. The current-voltage relationship of the nexal membrane was found to be linear, revealing a resistance r_n of 2–4 M. r_n was insensitive to the sarcolemmal membrane potential (range:–90 to +30 mV), and exerted no time-dependent gating behavior (range: 0.1 to 10 s). Lowering pH_i yielded a small increase in r_n. Vigorous elevations in [Ca²⁺]_i gave rise to an increase in r_n which was associated with a cell shortening. Uncoupling caused by aliphatic alcohols or halothane did not produce cell shortening. Cell pairs were also used to study action potential transfer.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Ionic mechanisms in pancreatic β cell signaling

The function and survival of pancreatic β cells critically rely on complex electrical signaling systems composed of a series of ionic events, namely fluxes of K⁺, Na⁺, Ca²⁺ and Cl^? across the β cell membranes. These electrical signaling systems not only sense events occurring in the extracellular space and intracellular milieu of pancreatic islet cells, but also control different β cell activities, most notably glucose-stimulated insulin secretion. Three major ion fluxes including K⁺ efflux through ATP-sensitive K⁺ (K_ATP) channels, the voltage-gated Ca²⁺ (Ca_V) channel-mediated Ca²⁺ influx and K⁺ efflux through voltage-gated K⁺ (K_V) channels operate in the β cell. These ion fluxes set the resting membrane potential and the shape, rate and pattern of firing of action potentials under different metabolic conditions. The K_ATP channel-mediated K⁺ efflux determines the resting membrane potential and keeps the excitability of the β cell at low levels. Ca²⁺ influx through Ca_V1 channels, a major type of β cell Ca_V channels, causes the upstroke or depolarization phase of the action potential and regulates a wide range of β cell functions including the most elementary β cell function, insulin secretion. K⁺ efflux mediated by K_V2.1 delayed rectifier K⁺ channels, a predominant form of β cell K_V channels, brings about the downstroke or repolarization phase of the action potential, which acts as a brake for insulin secretion owing to shutting down the Ca_V channel-mediated Ca²⁺ entry. These three ion channel-mediated ion fluxes are the most important ionic events in β cell signaling. This review concisely discusses various ionic mechanisms in β cell signaling and highlights K_ATP channel-, Ca_V1 channel- and K_V2.1 channel-mediated ion fluxes.     

Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe

Effects of extracellular magnesium ions ([Mg²⁺]_o ) on intracellular free Mg²⁺ ([Mg²⁺]_i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg²⁺ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg²⁺]_o , [Mg²⁺]_i in yeast cells was 0.91±0.08 mM. Elevation of [Mg²⁺]_o to 1.97 mM induced rapid (within 5 min) increments in [Mg²⁺]_i (2.18±0.11 mM). Lowering [Mg²⁺]_o to 0.06 mM, however, exerted no significant effects on [Mg²⁺]_i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg²⁺]_o used, the subcellular distribution of [Mg²⁺]_i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg²⁺] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg²⁺]_i when placed in 1.97 mM [Mg²⁺]_o . We conclude that [Mg²⁺]_i in fission yeast is maintained at a physiologic level when [Mg²⁺]_o is low, but intracellular free Mg²⁺ rapidly rises when [Mg²⁺]_o is elevated. Like most eukaryotic cells, yeast may have a Mg²⁺ transport system(s) which functions to maintain gradients of Mg²⁺ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996     

Changes in intracellular pH and cell volume during the early phase of DMSO-induced differentiation of friend erythroleukemia cells

Summary Changes in intracellular pH and water volume were measured after treatment of Friend erythroleukemia cells with 1.5% DMSO. It was found that a continuous decrease in pH_i occurred, beginning 1 h after induction and a decline in pH_i of 0.18 was measured after 9 h. In addition a decline in cellular water volume, of 12% only 15 min after induction, and 23% after 9 h, was observed.11 December 1986Acknowlegments. This work was supported by the Deutsche Forschungsgemeinschaft.     

Untersuchungen an Ionenaustauschmodellen zum Verständnis der Physiologie biogener Amine

Summary A synthetic ion exchanger containingR-PO(OH)₂ as active groups was shown to retain histamine and other basic substances selectively and, in most cases, with marked preference over the other cations (Na⁺, H⁺) present in the system. The results are taken to indicate that similar exchange reactions may occur on biogenic polyelectrolytes bearing similar active groups.     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

Differences in membrane ion transport between hepatocytes from the periportal and the pericentral areas of the liver lobule

We studied the Na⁺/K⁺ pump, Na⁺/K⁺ ATPase activity, and oxygen consumption (QO₂) in hepatocytes isolated from the periportal (PH) and pericentral (CH) regions of the liver lobule, to provide an insight into the functional properties of these cells. Na⁺/K⁺ pump activity was determined using⁸⁶Rb⁺ (a functional analog of K⁺) and ouabain, a specific inhibitor of this transport system. Our results indicate the the Na⁺/K⁺, pump and Na⁺/K⁺ ATPase activity are significantly lower in CH than in PH, although basal ouabain-sensitive (OS) QO₂ was negligible in both of these cell preparations. However, OSQO₂ was significantly lower in CH than in PH when the Na⁺/K⁺ pump was activated using the ionophore nystatin in a Na⁺-containing medium. These results indicate that the differences in membrane ion transport exist between hepatocytes from different locations of the liver lobule.     

Flippases: still more questions than answers

Our understanding of flippase-mediated lipid translocation and membrane vesiculation, and the involvement of P-type ATPases in these processes is just beginning to emerge. The results obtained so far demonstrate significant complexity within this field and point to major tasks for future research. Most importantly, biochemical characterization of P₄-ATPases is required in order to clarify whether these transporters indeed are capable of catalyzing transmembrane phospholipid flipping. The β-subunit of P₄-ATPases shows unexpected similarities between the β- and γ-subunits of the Na⁺/K⁺-ATPase. It is likely that these proteins provide a similar solution to similar problems, and might have adopted similar structures to accomplish these tasks. No P₄-ATPases have been identified in the endoplasmic reticulum and it remains an intriguing possibility that, in this compartment, P_5A-ATPases are functional homologues of P₄-ATPases. Received 19 June 2008; received after revision 31 July 2008; accepted 15 August 2008