Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed‐batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg^∑1) resulting from feeding of nutrients may enhance specific product formation rates (q_p). As an improved ATP supply was anticipated to enhance q_p this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg^‐1). Hyperosmotic culture conditions caused a ～2.6‐fold increase of specific ATP formation rates together with a ～1.7‐fold rise in cytosolic and mitochondrial ATP‐pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR‐132‐3p, miR‐132‐5p, miR‐182, miR‐183, miR‐194, miR‐215‐3p, miR‐215‐5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR‐183 may be the most promising target to enhance q_p by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.     

Interaction between thapsigargin and ATP4− in the regulation of the intracellular calcium in rat submandibular glands

Rat submandibular glands were digested with crude collagenase, and the intracellular calcium concentration of the cellular suspension was measured using fura-2. In the absence of extracellular magnesium and calcium ([Ca²⁺]_o), ATP had no effect; the response to ATP peaked at 1–2.5 mM [Ca²⁺]_o and was inhibited at 5 mM. One millimolar (mM) extracellular ATP did not increase the leak of LDH or fura-2; 10 m?M Coomassie brilliant blue G specifically inhibited the effect of ATP on [Ca²⁺]_in. Depleting intracellular calcium pools with thapsigargin did not affect the response to ATP. Using a Ca²⁺-free/Ca²⁺ reintroduction protocol, it was shown that ATP and thapsigargin increase the uptake of extracellular calcium. The effect of the two agonists was synergistic. Removal of extracellular sodium inhibited the effect of carbachol on [Ca²⁺]_in and the calcium uptake but potentiated the response to ATP. These results suggest that, after binding to purinergic receptors, extracellular ATP^4- increases [Ca²⁺]_in. ATP^4- does not mobilize thapsigargin-sensitive intracellular calcium pools (among which is the IP₃-sensitive calcium pool) but stimulates the uptake of extracellular calcium by a mechanism inhibited by extracellular sodium, probably by opening a nonselective cation channel. © 1994 Wiley-Liss, Inc.     

Intracellular sodium homeostasis in relation to the effective free-energy change of ATP hydrolysis: studies of crayfish muscle fibers

We investigated the impact of reductions in the effective free-energy change of ATP hydrolysis (dG/d_ATP) on intracellular sodium homeostasis in bundles of fibers from the abdominal extensor muscle of the crayfish Procambarus clarkii. ³¹P nuclear magnetic resonance (NMR) spectroscopy was used to monitor high-energy phosphate levels and intracellular pH while interleaved ²³Na-NMR spectra were acquired to monitor changes in sodium. Previous work has shown that the bulk of intracellular Na⁺ is NMR visible (see Ivanics et al. 1994). The ²³Na-NMR spectra were diffusion-weighted which effectively filtered out signal contributions from extracellular sodium. The efficacy of this procedure was validated using a relatively non-toxic chemical shift reagent which allowed resolution of extracellular and intracellular Na⁺ signals. Metabolic inhibition (cyanide/iodoacetate) produced pronounced reductions in dG/d_ATP coincident with dramatic increases in intracellular Na⁺ levels ([Na⁺]_i). However, the increases in [Na⁺]_i occurred at dG/d_ATP values well above the threshold value of −46 kJ · mol⁻¹ required by the existing Na⁺ gradient and the membrane potential. These results suggest that the global dG/d_ATP value may not reflect the dG/d_ATP value in the vicinity of the pump. Alternatively, other factors, including low molecular modulators of Na⁺, K⁺-ATPase activity, may be important in this context. Accepted: 15 May 1997     

Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study

The effect of individual environmental conditions (pH, pO₂, temperature, salinity, concentration of ethanol, propanol, tryptone and yeast extract) on the specific growth rate as well as ethanol and glycerol production rate of Saccharomyces cerevisiae S288C was mapped during the fermentative growth in aerobic auxo-accelerostat cultures. The obtained steady-state values of the glycerol to ethanol formation ratio (0.1 mol mol⁻¹) corresponding to those predicted from the stoichiometric model of fermentative yeast growth showed that the complete repression of respiration was obtained in auxostat culture and that the model is suitable for calculation of Y _ATP and Q _ATP values for the aerobic fermentative growth. Smooth decrease in the culture pH and dissolved oxygen concentration (pO₂) down to the critical values of 2.3 and 0.8%, respectively, resulted in decrease in growth yield (Y _ATP) and specific growth rate, however the specific ATP production rate (Q _ATP) stayed almost constant. Increase in the concentration of biomass (>0.8 g dwt l⁻¹), propanol (>2 g l⁻¹) or NaCl (>15 g l⁻¹) lead at first to the decrease in the specific growth rate and Q _ATP, while Y _ATP was affected only at higher concentrations. The observed decrease in Q _ATP was caused by indirect rather than direct inhibition of glycolysis. The increase in tryptone concentration resulted in an increase in the specific growth rate from 0.44 to 0.62 h⁻¹ and Y _ATP from 12.5 to 18.5 mol ATP g dwt⁻¹. This study demonstrates that the auxo-accelerostat method, besides being an efficient tool for obtaining the culture characteristics, provides also decent conditions for the experiments elucidating the control mechanisms of cell growth.     

Mechanism of ATP-sensitive K Channel Inhibition by Sulfhydryl Modification

ATP-sensitive potassium (K_ATP) channels are reversibly inhibited by intracellular ATP. Agents that interact with sulfhydryl moieties produce an irreversible inhibition of K_ATP channel activity when applied to the intracellular membrane surface. ATP appears to protect against this effect, suggesting that the cysteine residue with which thiol reagents interact may either lie within the ATP-binding site or be inaccessible when the channel is closed. We have examined the interaction of the membrane-impermeant thiol-reactive agent p-chloromercuriphenylsulphonate (pCMPS) with the cloned β cell K_ATP channel. This channel comprises the pore-forming Kir6.2 and regulatory SUR1 subunits. We show that the cysteine residue involved in channel inhibition by pCMPS resides on the Kir6.2 subunit and is located at position 42, which lies within the NH₂ terminus of the protein. Although ATP protects against the effects of pCMPS, the ATP sensitivity of the K_ATP channel was unchanged by mutation of C42 to either valine (V) or alanine (A), suggesting that ATP does not interact directly with this residue. These results are consistent with the idea that C42 is inaccessible to the intracellular solution, and thereby protected from interaction with pCMPS when the channel is closed by ATP. We also observed that the C42A mutation does not affect the ability of SUR1 to endow Kir6.2 with diazoxide sensitivity, and reduces, but does not prevent, the effects of MgADP and tolbutamide, which are mediated via SUR1. The Kir6.2-C42A (or V) mutant channel may provide a suitable background for cysteine-scanning mutagenesis studies.     

Nicotinamide‐rich diet improves physical endurance by up‐regulating SUR2A in the heart

SUR2A is an ATP‐binding protein that serves as a regulatory subunit of cardioprotective ATP‐sensitive K⁺ (K_ATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled K_ATP channels, we have suggested that nicotinamide‐rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide‐rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild‐type mice on nicotinamide‐rich diet. The experiments focused on action membrane potential and intracellular Ca²⁺ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal K_ATP channels and steady Ca²⁺ levels in cardiomyocytes in response to β‐adrenergic stimulation. In contrast, the same challenge in the wild‐type was characterized by a lack of the channel activation and rise in intracellular Ca²⁺. Nicotinamide‐rich diet was ineffective to increase physical endurance in mice lacking K_ATP channels. This study has shown that nicotinamide‐rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide‐rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.     

KATP channels process nucleotide signals in muscle thermogenic response

Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K⁺ (K_ATP) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, K_ATP channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how K_ATP channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the K_ATP channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with K_ATP channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal K_ATP channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of K_ATP channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of K_ATP channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders.     

AMPK modulates glucose-sensing in insulin-secreting cells by altered phosphotransfer to KATP channels

Glucose-sensing (GS) behaviour in pancreatic β-cells is dependent on ATP-sensitive K⁺ channel (K_ATP) activity, which is controlled by the relative levels of the K_ATP ligands ATP and ADP, responsible for closing and opening K_ATP, respectively. However, the mechanism by which β-cells transfer energy status from mitochondria to K_ATP, and hence to altered electrical excitability and insulin secretion, is presently unclear. Recent work has demonstrated a critical role for AMP-activated protein kinase (AMPK) in GS behaviour of cells. Electrophysiological recordings, coupled with measurements of gene and protein expression were made from rat insulinoma cells to investigate whether AMPK activity regulates this energy transfer process. Using the whole-cell recording configuration with sufficient intracellular ATP to keep K_ATP closed, raised AMPK activity induced GS electrical behaviour. This effect was prevented by the AMPK inhibitor, compound C and required a phosphotransfer process. Indeed, high levels of intracellular phosphocreatine or the presence of the adenylate kinase (AK) inhibitor AP₅A blocked this action of AMPK. Using conditions that maximised AMPK-induced K_ATP opening, there was a significant increase in AK1, AK2 and UCP2 mRNA expression. Thus we propose that K_ATP opening in response to lowered glucose concentration requires AMPK activity, perhaps in concert with increased AK and UCP2 to enable mitochondrial-derived ADP signals to be transferred to plasma membrane K_ATP by phosphotransfer cascades.     

Differential effects of propofol and isoflurane on glucose utilization and insulin secretion

AimsVolatile anesthetics, such as isoflurane, reverse glucose-induced inhibition of pancreatic adenosine triphosphate-sensitive potassium (K_ATP) channel activity, resulting in reduced insulin secretion and impaired glucose tolerance. No previous studies have investigated the effects of intravenous anesthetics, such as propofol, on pancreatic K_ATP channels. We investigated the cellular mechanisms underlying the effects of isoflurane and propofol on pancreatic K_ATP channels and insulin secretion.Main methodsIntravenous glucose tolerance tests (IVGTT) were performed on male rabbits. Pancreatic islets were isolated from male rats and used for a perifusion study, measurement of intracellular ATP concentration ([ATP]_i), and patch clamp experiments.Key findingsGlucose stimulus significantly increased insulin secretion during propofol anesthesia, but not isoflurane anesthesia, in IVGTT study. In perifusion experiments, both islets exposed to propofol and control islets not exposed to anesthetic had a biphasic insulin secretory response to a high dose of glucose. However, isoflurane markedly inhibited glucose-induced insulin secretion. In a patch clamp study, the relationship between ATP concentration and channel activity could be fitted by the Hill equation with a half-maximal inhibition of 22.4, 15.8, and 218.8 μM in the absence of anesthetic, and with propofol, and isoflurane, respectively. [ATP]_i and single K_ATP channel conductance did not differ in islets exposed to isoflurane or propofol.SignificanceOur results indicate that isoflurane, but not propofol, decreases the ATP sensitivity of K_ATP channels and impairs glucose-stimulated insulin release. These differential actions of isoflurane and propofol on ATP sensitivity may explain the differential effects of isoflurane and propofol on insulin release.     

Kir6.2 Channel Gating by Intracellular Protons: Subunit Stoichiometry for Ligand Binding and Channel Gating

The adenosine triphosphate-sensitive K⁺ (K_ATP) channels are gated by several metabolites, whereas the gating mechanism remains unclear. Kir6.2, a pore-forming subunit of the K_ATP channels, has all machineries for ligand binding and channel gating. In Kir6.2, His175 is the protonation site and Thr71 and Cys166 are involved in channel gating. Here, we show how individual subunits act in proton binding and channel gating by selectively disrupting functional subunits using these residues. All homomeric dimers and tetramers showed pH sensitivity similar to the monomeric channels. Concatenated construction of wild type with disrupted subunits revealed that none of these residues had a dominant-negative effect on the proton-dependent channel gating. Subunit action in proton binding was almost identical to that for channel gating involving Cys166, suggesting a one-to-one coupling from the C terminus to the M2 helix. This was significantly different from the effect of T71Y heteromultimers, suggesting distinct contributions of M1 and M2 helices to channel gating. Subunits underwent concerted rather than independent action. Two wild-type subunits appeared to act as a functional dimer in both cis and trans configurations. The understanding of K_ATP channel gating by intracellular pH has a profound impact on cellular responses to metabolic stress as a significant drop in intracellular pH is more frequently seen under a number of physiological and pathophysiological conditions than a sole decrease in intracellular ATP levels. Runping Wang, Junda Su contributed equally to this work.     

The uncoupling protein and the potassium channel are activated by hyperosmotic stress in mitochondria from durum wheat seedlings

The plant uncoupling mitochondrial protein (PUMP) and the plant mitochondrial potassium channel (PmitoK_ATP) are two recently discovered energy‐dissipating systems present in plant mitochondria, which may play a role as defence systems under environmental stress. To verify whether hyperosmotic stress affects the two dissipating systems in durum wheat (Triticum durum Desf.), their functioning was studied in early etiolated seedlings maintained under moderate and severe salt (NaCl) and osmotic (mannitol) stress. As measures of mitochondrial stress mitochondrial integrity, membrane potential maintenance and oxygen uptake coupled with ATP synthesis during succinate and proline oxidation were investigated. Both PUMP and PmitoK_ATP were activated under stress conditions. Activation was clearly evident even under moderate stress when proline oxidation was inhibited, although mitochondrial integrity and succinate oxidation were still unaffected. Under severe stress, which significantly affected all the tested indicators of mitochondrial integrity and functionality, PUMP and PmitoK_ATP activation was further enhanced. Interestingly, both systems were activated by reactive oxygen species and were able to control mitochondrial superoxide anion production. These results suggest that PUMP and PmitoK_ATP serve as early antioxidant defence systems in response to hyperosmotic stress and that they are involved in a prolonged response to stress.     

M-LDH physically associated with sarcolemmal KATP channels mediates cytoprotection in heart embryonic H9C2 cells

Muscle form of lactate dehydrogenase (M-LDH) physically associate with K_ATP channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K⁺ (K_ATP) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (ΔM-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the K_ATP channel protein complex. The K⁺ current was increased in wild type cells, but not in cells expressing ΔM-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10 mM). At the same time, the outcome of chemical hypoxia was much worse in ΔM-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with ΔM-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes K_ATP channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing ΔM-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal K_ATP channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival.     

Regulation of intracellular H+ balance in Zymomonas mobilis 113 during the shift from anaerobic to aerobic conditions

Summary The energetics, enzyme activities and end-product synthesis of Zymomonas mobilis 113 in continuous culture were studied after the shift from an anaerobic to an aerobic environment. Aeration diminished ethanol yield and lactic acid concentration, but increased glucose consumption rate and production of acetic acid. After the shift to aerobic conditions reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase activity was stimulated. Washed cell suspensions consumed oxygen with glucose, lactate and ethanol as substrates. The aerobic Z. mobilis 113 regulated their intracellular redox balance by production and reoxidation of the end products, coupled with the formation of NAD(P)H. An increase in transmembrane pH gradient (pH) and a decrease in intracellular ATP concentration were observed after the shift to aerobic conditions. At low medium redox potential (Eh) values the H⁺ balance was regulated in an energy-independent way via end-product excretion. Under aerobic conditions this was supplemented by ATP-dependent H⁺ excretion by the membrane H⁺-ATPase.Abbreviations D dilution rate (h^-1) - S ₀ initial glucose concentration (g/l) - Y _x/s growth yield (g/mol) - Y _p/s product yield (g/g) - q _s specific rate of substrate utilization (g/g per hour) - q _p specific rate of ethanol formation (g/g per hour) - qo ₂ specific rate of CO₂ production (mmol/g per hour) - specific growth rate (h^-1) - X dry biomass concentration (g/l) - Eh redox potential of culture medium (mV) - pH transmembrane pH gradient (pH units) - pH_in intracellular pH - SASE sum of activities of specific enmymes of Entner-Doudoroff pathway     

The cystathionine‐β‐synthase domains on the guanosine 5′’‐monophosphate reductase and inosine 5′‐monophosphate dehydrogenase enzymes from Leishmania regulate enzymatic activity in response to guanylate and adenylate nucleotide levels

The Leishmania guanosine 5′‐monophosphate reductase (GMPR) and inosine 5′‐monophosphate dehydrogenase (IMPDH) are purine metabolic enzymes that function maintaining the cellular adenylate and guanylate nucleotide. Interestingly, both enzymes contain a cystathionine‐β‐synthase domain (CBS). To investigate this metabolic regulation, the Leishmania GMPR was cloned and shown to be sufficient to complement the guaC (GMPR), but not the guaB (IMPDH), mutation in Escherichia coli. Kinetic studies confirmed that the Leishmania GMPR catalyzed a strict NADPH‐dependent reductive deamination of GMP to produce IMP. Addition of GTP or high levels of GMP induced a marked increase in activity without altering the K_m values for the substrates. In contrast, the binding of ATP decreased the GMPR activity and increased the GMP K_m value 10‐fold. These kinetic changes were correlated with changes in the GMPR quaternary structure, induced by the binding of GMP, GTP, or ATP to the GMPR CBS domain. The capacity of these CBS domains to mediate the catalytic activity of the IMPDH and GMPR provides a regulatory mechanism for balancing the intracellular adenylate and guanylate pools.     

Nicorandil regulates the macrophage skewing and ameliorates myofibroblasts by inhibition of RhoA/Rho‐kinase signalling in infarcted rats

We have demonstrated that ATP‐sensitive potassium (K_ATP) channel agonists attenuated fibrosis; however, the mechanism remained unclear. Since RhoA has been identified as a mediator of cardiac fibrosis, we sought to determine whether the anti‐fibrotic effects of K_ATP channel agonists were mediated via regulating macrophage phenotype and fibroblast differentiation by a RhoA/RhoA‐kinase‐dependent pathway. Wistar male rats after induction of myocardial infarction were randomized to either vehicle, nicorandil, an antagonist of K_ATP channel glibenclamide, an antagonist of ROCK fasudil, or a combination of nicorandil and glibenclamide or fasudil and glibenclamide starting 24 hrs after infarction. There were similar infarct sizes among the infarcted groups. At day 3 after infarction, post‐infarction was associated with increased RhoA/ROCK activation, which can be inhibited by administering nicorandil. Nicorandil significantly increased myocardial IL‐10 levels and the percentage of regulatory M2 macrophages assessed by immunohistochemical staining, Western blot, and RT‐PCR compared with vehicle. An IL‐10 receptor antibody increased myofibroblast infiltration compared with nicorandil alone. At day 28 after infarction, nicorandil was associated with attenuated cardiac fibrosis. These effects of nicorandil were functionally translated in improved echocardiographically derived cardiac performance. Fasudil showed similarly increased expression of M2 macrophages as nicorandil. The beneficial effects of nicorandil on fibroblast differentiation were blocked by adding glibenclamide. However, glibenclamide cannot abolish the attenuated fibrosis of fasudil, implying that RhoA/RhoA‐kinase is a downstream effector of K_ATP channel activation. Nicorandil polarized macrophages into M2 phenotype by inhibiting RhoA/RhoA‐kinase pathway, which leads to attenuated myofibroblast‐induced cardiac fibrosis after myocardial infarction.     

Physiological responses of Zygosaccharomyces rouxii to osmotic stress

When cell suspensions of Zygosaccharomyces rouxii were subjected to osmotic shock with NaCl, the cell volume decreased sharply and plasmolysis was observed. The cell subsequently recovered and volumes similar to those of cells growing at the respective water activity (a_w) values were found. Cycloheximide prevented cell recovery, indicating the involvement of protein synthesis in the recovery process. The intracellular glycerol concentration of Z. rouxii incubated in the presence of [¹⁴C]glycerol increased from 13 to 96 mmol/l during the initial 20 min after an upshock from 0.998 a_w to 0.96 a_w. All the intracellular glycerol was labelled and therefore derived from the medium. Labelled glycerol was subsequently utilized and replaced by unlabelled glycerol produced by the cell within 90 min. The initial increase in glycerol concentration following the upshock was confirmed by ¹³C-nuclear magnetic resonance (NMR) spectroscopic studies of cell extracts. The combined dihydroxyacetone and dihydroxyacetone phosphate concentrations fluctuated during this period, whereas glycerol-3-phosphate initially increased and then remained constant. This indicates that the production of glycerol is regulated. Decreases in ATP and polyphosphate levels were observed following osmotic upshock and may reflect a greater demand for ATP during the period of adjustment to decreased a_w. The changes in cell volume and in ATP concentration following osmotic upshock may serve as osmoregulatory signals in Z. rouxii, as suggested previously for other microorganisms. Correspondence to: S. G. Kilian     

Opening of Cardiac Sarcolemmal KATP Channels by Dinitrophenol Separate from Metabolic Inhibition

Opening of ATP-sensitive K⁺ (K_ATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 μm) can induce opening of cardiac K_ATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1–2.5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, τ_open: 4.8 msec; fast closed time constant, τ_closed(f): 0.33 msec) characteristic of K_ATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open K_ATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of K_ATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced K_ATP channel opening should be reevaluated. Received: 10 September 1996/Revised: 27 December 1996     

Photoinhibition of photosystem II in vivo is preceded by down-regulation through light-induced acidification of the lumen: Consequences for the mechanism of photoinhibition in vivo

The mechanism of photoinhibition of photosystem II (PSII) was studied in intact leaf discs of Spinacia oleracea L. and detached leaves of Vigna unguiculata L. The leaf material was exposed to different photon flux densities (PFDs) for 100 min, while non-photochemical (q_N) and photochemical quenching (q_p) of chlorophyll fluorescence were monitored. The ‘energy’ and redox state of PSII were manipulated quite independently of the PFD by application of different temperatures (5–20° C), [CO₂] and [O₂] at different PFDs. A linear or curvilinear relationship between q_p and photoinhibition of PSII was observed. When [CO₂] and [O₂] were both low (30 μl · l^?1 and 2%, respectively), PSII was less susceptible at a given q_p than at ambient or higher [CO₂] and photoinhibition became only substantial when q_p decreased below 0.3. When high levels of energy-dependent quenching (q_E) (between 0.6 and 0.8) were reached, a further increase of the PFD or a further decrease of the metabolic demand for ATP and NADPH led to a shift from q_E to photoinhibitory quenching (q_I). This shift indicated that photoinhibition was preceded by down-regulation through light-induced acidification of the lumen. We propose that photoinhibition took place in the centers down-regulated by q_E. The shift from q_E to q_I occurred concomitant with q_P decreasing to zero. The results clearly show that photoinhibition does not primarily depend on the photon density in the antenna, but that photoinhibition depends on the energy state of the membrane in combination with the redox balance of PSII. The results are discussed with regard to the mechanism of photoinhibition of PSII, considering, in particular, effects of light-induced acidification on the donor side of PSII. Interestingly, cold-acclimation of spinach leaves did not significantly affect the relationship between q_P, q_E and photoinhibition of PSII at low temperature.