Spin-probes designed for measuring the intrathylakoid pH in chloroplasts

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pH_in established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pH_out = 7.8, we found that pH_in ≈ 5.4-5.7 in the state of photosynthetic control, and pH_in ≈ 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8-2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H⁺/ATP is n ≥ 4-4.7.     

Kinetic analysis of nonphotochemical quenching of chlorophyll fluorescence. 1. Isolated chloroplasts

Nonphotochemical quenching of chlorophyll fluorescence in plants is indicative of a process that dissipates excess excitation energy from the light-harvesting antenna of photosystem II. The major fraction of quenching is obligatorily dependent upon the thylakoid DeltapH and is regulated by the de-epoxidation state of the xanthophyll cycle carotenoids associated with the light-harvesting complexes. Basic principles of enzyme kinetics have been used to investigate this process in isolated chloroplasts. The extent of quenching was titrated against the estimated thylakoid lumen pH, and a sigmoidal relationship was obtained with a Hill coefficient of 4.5 and a pK of 4.7. Upon de-epoxidation, these parameters changed to 1.6 and 5.7, respectively. Antimycin A suppressed quenching, increasing the Hill coefficient and reducing the pK. The rate of induction of quenching fitted second-order kinetics with respect to illumination time, and the rate constant was dependent upon the DeltapH, the de-epoxidation state, the presence of antimycin, and also the presence of dibucaine, a quenching enhancer. All these data are consistent with the notion that quenching is caused by a conformational transition in a chloroplast thylakoid protein; this transition shows cooperativity with respect to proton binding, and is controlled by de-epoxidation state and various exogenous reagents.     

In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana

Among the protein translocation pathways of the thylakoid membrane in chloroplasts, the DeltapH/TAT pathway is unique in several aspects. In vitro transport assays with isolated chloroplasts or thylakoids have defined the trans-thylakoidal proton gradient as the sole requirement for effecting transport. From these studies, evidence has also accumulated indicating that, in contrast to the remaining protein transport pathways present in the thylakoid membrane, the DeltapH/TAT pathway is able to mediate the transport of folded proteins. The present work has established a novel approach to demonstrate the transport of folded proteins by this pathway in vivo. For this purpose, Arabidopsis thaliana plants were stably transformed with gene constructs expressing enhanced green fluorescent protein (EGFP) alone or fused to the transit peptides of different chloroplast proteins under the control of the 35S CAMV promoter. The intracellular and intraorganellar distribution of EGFP in the resulting transformants showed that while all the chloroplast transit peptides efficiently mediated the transport of EGFP into plastids, only those specific for the DeltapH/TAT pathway were able to direct the protein into the thylakoid lumen as well. This could be demonstrated both by fluorescence and immunoelectron microscopy. Analysis of isolated and fractionated chloroplasts using western blot and spectrofluorometric assays confirmed the presence of folded EGFP solely within the thylakoid lumen of these lines. These results strongly suggest that the protein adopts a folded state in the chloroplast stroma and thus, can only be translocated further into the chloroplast lumen by the DeltapH/TAT pathway.     

Comparison of DeltapH- and Delta***φ***-driven ATP synthesis catalyzed by the H(+)-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes.

The H(+)-ATPases from Escherichia coli, EF(0)F(1), and from chloroplasts, CF(0)F(1), were reconstituted in liposomes from phosphatidylcholine/phosphatidic acid. The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential and the initial rate of ATP synthesis was measured as a function of the transmembrane pH difference, DeltapH, and the electric potential difference, Deltaφ. With EF(0)F(1), a rate of 80 s(-1) is observed at DeltapH=4.1 and Deltaφ approximately 140 mV. The rate decreases sigmoidally with Deltaφ and at Deltaφ approximately 0 mV, the rate is about 1 s(-1) although DeltapH is still 4.1. Under the same conditions with CF(0)F(1), a rate of 280 s(-1) is observed which decreases to 190 s(-1) when Deltaφ is abolished, i.e. ATP synthesis catalyzed by EF(0)F(1) and CF(0)F(1) depends in a different way on DeltapH and Deltaφ. EF(0)F(1)-catalyzed ATP synthesis was measured as a function of DeltapH at a constant Deltaφ. The rate depends sigmoidally on DeltapH reaching a maximal rate which cannot be further increased by increasing DeltapH. However, this maximal rate depends on Deltaφ, i.e. DeltapH and Deltaφ are not kinetically equivalent in driving ATP synthesis. We assume that EF(0)F(1) must be converted into a metastable, active state before it catalyzes proton transport-coupled ATP synthesis. For EF(0)F(1), this activation step depends only on Deltaφ, whereas for CF(0)F(1), the activation depends on DeltapH and Deltaφ.     

Transport of Proteins into the Thylakoid Lumen--Stromal Processing and Energy Requirements for the Import of the Precursor to the 23-kDa Protein of PS II

Transport of the precursor to the 23-kDa protein of photosystemII was examined by incubation of the precursor with isolatedintact chloroplasts in the presence of ATP in darkness. An intermediary-sizedform was accumulated in the stroma at 0.1–1 mM ATP. Athigher concentrations of ATP (3.2–10 mM), the precursorwas imported into the thylakoid lumen and processed to the matureform. The precursor was not imported even as far as the stromain the absence of ATP. The intermediary-sized form that accumulatedat low concentrations of ATP was imported into the thylakoidlumen and processed to the mature form when chloroplasts weresubsequently incubated in the light. These observations indicatethat the accumulated intermediary-sized form was suitable forfurther translocation and that the intermediary-sized form isa transport intermediate that occurs under natural conditions.Import of the protein into the thylakoid lumen, which was observedat the higher concentrations of ATP, was inhibited by the additionof nigericin or carbonylcyanide m-chlorophenyl hydrazine. Theeffects of these ionophores suggests that the translocationof the protein across thylakoid membrane requires a proton gradientacross the membrane. The results together show that the proteinis imported from the cytosol into the thylakoid lumen in discretesteps: ATP-driven translocation across envelope membranes, stromalprocessing to the intermediate, translocation of the intermediateacross the thylakoid membrane and final processing to the matureprotein within the thylakoids. (Received June 3, 1992; Accepted December 17, 1992)     

Fluorescent probes for non-invasive bioenergetic studies of whole cyanobacterial cells.

Fluorescent DeltapH and DeltaPsi indicators have been screened for the non-invasive monitoring of bioenergetic processes in whole cells of the cyanobacterium Synechocystis sp. PCC 6803. Acridine yellow and Acridine orange proved to be the best DeltapH indicators for the investigation of thylakoid and cytoplasmic membrane energization: While Acridine yellow indicated only cytosolic energization, Acridine orange showed signals from both the thylakoid lumen and the cytosol that could be separated kinetically. Both indicators were applied successfully to monitor cellular energetics, such as the interplay of linear and cyclic photosynthetic electron transport, osmotic adaptation and solute transport across the cytoplasmic membrane. In contrast, useful membrane potential indicators were more difficult to find, with Di-4-ANEPPS and Brilliant cresyl blue being the only promising candidates for further studies. Finally, Acridine yellow and Acridine orange could also be applied successfully for the thermophilic cyanobacterium Synechococcus elongatus. Different from Synechocystis sp. PCC 6803, where both respiration and ATP hydrolysis could be utilized for cytoplasmic membrane energization, proton extrusion at the cytoplasmic membrane in Synechococcus elongatus was preferentially driven by ATP hydrolysis.     

Magnetic resonance study of the transmembrane nitrite diffusion.

Nitrite (NO(2)-), being a product of metabolism of both nitric oxide (NO(*)) and nitrate (NO(3)-), can accumulate in tissues and regenerate NO() by several mechanisms. The effect of NO(2)- on ischemia/reperfusion injury was also reported. Nevertheless, the mechanisms of intracellular NO(2)- accumulation are poorly understood. We suggested significant role of nitrite penetration through biological membranes in the form of undissociated nitrous acid (HNO(2)). This hypothesis has been tested using large unilamellar phosphatidylcholine liposomes and several spectroscopic techniques. HNO(2) transport across the phospholipid bilayer of liposomes facilitates proton transfer resulting in intraliposomal acidification, which was measured using pH-sensitive probes. NO(2)(-)-mediated intraliposomal acidification was confirmed by EPR spectroscopy using membrane-impermeable pH-sensitive nitroxide, AMC (2,2,5,5-tetramethyl-1-yloxy-2,5-dihydro-1H-imidazol-3-ium-4-yl)-aminomethanesulfonic acid (pK 5.25), and by (31)P NMR spectroscopy using inorganic phosphate (pK 6.9). Nitrite accumulates inside liposomes in concentration exceeding its concentration in the bulk solution, when initial transmembrane pH gradient (alkaline inside) is applied. Intraliposomal accumulation of NO(2)- was observed by direct measurement using chemiluminescence technique. Perfusion of isolated rat hearts with buffer containing 4 microM NO(2)- was performed. The nitrite concentrations in the effluent and in the tissue, measured after 1 min perfusion, were close, supporting fast penetration of the nitrite through the tissue. Measurements of the nitrite/nitrate showed that total concentration of NO(x) in myocardium increased from initial 7.8 to 24.7 microM after nitrite perfusion. Physiological significance of passive transmembrane transport of NO(2)- and its coupling with intraliposomal acidification are discussed.     

Full-length plastocyanin precursor is translocated across isolated thylakoid membranes

In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.     

Structure of Aurasperone C

Aurasperone C (III) shows properties closely related to those of aurasperone B (II) and gave dianhydro compound (V) on hydrochloric acid treatment. Partial methylation of (V) with methyl iodide afforded a monomethyl ether identical with aurasperone A (I).

NMR studies, including solvent induced methoxyl shifts, indicate the structure of (III) to be 2,2′-dimethyl-2,2′,5,5′-,8-pentahydroxy-6,6′,8-trimethoxy-7,10′-bi[2,3-dihydro-4H-naphtho[2,3b]pyran-4-one], in which the 8-methoxyl of aurasperone B is replaced by a hydroxyl group.     

Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts

The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of [¹⁴C]5,5-dimethyl-2,4-oxazolidinedione and [¹⁴C] methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO₂ fixation by superoptimal medium Pi or by adding glyceraldehyde—an inhibitor of the Calvin cycle—leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific `low phosphate' effect could be partially reversed by adding oxaloacetate, which regenerates the electron acceptor NADP<sup>+</sup> and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N′-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N′-Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific `low phosphate' proton leak in the thylakoids, and its origin is discussed.     

Precursors bind to specific sites on thylakoid membranes prior to transport on the delta pH protein translocation system

The Delta pH pathway is one of two systems for protein transport to the thylakoid lumen. It is a novel transport system that requires only the thylakoidal DeltapH to power translocation. Several substrates of the Delta pH pathway, including the intermediate precursor form of OE17 (iOE17) and the truncated precursor form of OE17 (tOE17), were shown to bind to the membrane in the absence of the DeltapH and be transported into the lumen when the DeltapH was restored. Binding occurred without energy or soluble factors, and efficient transport from the bound state ( approximately 80-90%) required only the DeltapH. Binding is due to protein-protein interactions because protease pretreatment of thylakoids destroyed their binding capability. Precursors are bound to a specific site on the Delta pH pathway because binding was competed by saturating amounts of Delta pH pathway precursor proteins, but not by a Sec pathway precursor protein. These results suggested that precursor tOE17 binds to components of the Delta pathway translocation machinery. Hcf106 and Tha4 are two components of the Delta pH pathway machinery. Antibodies to Hcf106 or Tha4, when prebound to thylakoids, specifically inhibited precursor transport on the Delta pH pathway. However, only Hcf106 antibodies reduced the level of precursor binding. These results suggest that Hcf106 functions in early steps of the transport process.     

The haemolytic reactions of 1-acetyl-2-phenylhydrazine and hydrazine: A spin trapping study

Free radical production from the reaction of hydrazine and 1-acetyl-2-phenylhydrazine (AcPhHZ) with oxyhaemoglobin and with human red blood cells, has been observed by the electron spin resonance technique of spin trapping. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the free radical intermediates detected depended on the hydrazine derivative, oxyhaemoglobin and the oxyhaem/hydrazine derivative concentration ratio.

The reaction of hydrazine with oxyhaemoglobin in the presence of DMPO gave a nitroxide which was identified as a reduced dimer of DMPO. Whereas hydrazine-treated red blood cells, in the presence of DMPO, gave a nitroxide spin adduct which was identified as the hydroxyl radical spin adduct of DMPO, 5,5-dimethyl-1-pyrrolidino-1-oxyl (DMPO-OH).

The reaction of AcPhHZ with oxyhaemoglobin, in the presence of DMPO, gave DMPO-OH, the phenyl radical spin adduct of DMPO, 5,5-dimethyl-2-phenylpyrrolidino-1-oxyl (DMPO-Ph) and an oxidised derivative of DMPO, 5,5-dimethyl-2-pyrrolidone-1-oxyl (DMPOX). The amounts of DMPO-Ph, DMPO-OH and DMPOX observed depended on the 1-acetyl-2-phenyl-hydrazine/oxyhaemoglobin concentration ratio; DMPOX replaced DMPO-OH as the concentration of AcPhHZ was decreased. DMPOX production has been previously associated with the production of highly oxidised haem iron-oxygen intermediates. AcPhHZ treated red blood cells gave DMPO-Ph and DMPO-OH spin adducts in the presence of DMPO.

DMPO had little to no effect on the rate of oxygen consumption by oxyhaemoglobin with hydrazine and AcPhHZ. Moreover, the rate of oxyhaemoglobin oxidation induced by hydrazine, was not decreased by DMPO whereas the rate of oxyhaemoglobin oxidation induced by AcPhHZ was decreased approx. 40% by DMPO. DMPO (10 mM) gave a small decrease in haemolysis and lipid peroxidation induced by 1 mM hydrazine and AcPhHZ in a 1% suspension of red blood cells.     

Functional and topological aspects of pH-dependent regulation of electron and proton transport in chloroplasts in silico

In this work, we summarize results of computer simulation of electron and proton transport processes coupled to ATP synthesis in chloroplasts performed within the frames of a mathematical model developed as a system of differential equations for concentrations of electron carriers and hydrogen ion inside and outside the granal and stromal thylakoids. The model takes into account topological peculiarities and lateral heterogeneity of the chloroplast lamellar system. This allowed us to analyze the influence of restricted diffusion of protons inside small compartments of a chloroplast (e.g., in the narrow inter-thylakoid gap) on electron transport processes. The model adequately describes two modes of pH-dependent feedback control of electron transport associated with: (i) the acidification of the thylakoid lumen, which causes the slowing down of plastoquinol oxidation and stimulates an increase in dissipation of excess energy in PS2, and (ii) the alkalization of stroma, inducing the activation of the BBC (Bassham-Benson-Calvin) cycle and intensified consumption of ATP and NADPH. The influence of ATP on electron transport is mediated by modulation of the thylakoid membrane conductivity to protons through the ATP synthase complexes. We also analyze the contribution of alternative electron transport pathways to the maintenance of optimal balance between the energy donating and energy consuming stages of the light-induced photosynthetic processes.     

Effects of transmembrane gradients of bicarbonate and ammonium on junctional and nonjunctional membrane conductances in BHK cells

Weak acids are efficient blockers of gap-junctional conductance. It is generally accepted that intracellular acidification produced by weak acids fully accounts for the gap-junctional uncoupling. Protonation of the cytoplasmic portions of the channel-forming protein connexin is thought to lead to the conformational changes switching the channel from the open into the closed state. If this is the only mechanism of the weak-acid induced uncoupling, then the correlation between junctional conductance (Gj) and intracellular pH (pHin) should not depend on the means of intracellular acidification. We compared the responses of junctional conductance in BHK cells measured in double whole-cell experiments to the applied transmembrane concentration gradients of bicarbonate or ammonium. These treatments were to lower pHin in a predictable way according to the equations: pHin = pHout -lg[[HCO3]out/HCO3-]in) or pHin = PHout - lg[[NH4+]in[NH4+out), respectively. We found that the behavior of Gj depended on the substance used. At a 500-fold bicarbonate gradient (calculated pHin approximately 4.8) the cells remained coupled, while a 100- or 10-fold gradient of ammonium imposing pHin approximately 6.1 produced fast uncoupling. The responses of junctional conductance were often accompanied or preceded by changes of non-junctional membrane conductance. We suggest that the mechanisms of the weak acid/base-induced channel gating may contain an additional "lipophilic" component due to the presence of the non-dissociated form of the acid/base in cell membrane.     

Ion selectivity and activation of the M2 ion channel of influenza virus.

The influenza A virus-associated M2 ion channel is generally believed to function during uncoating of virions in infected cells. On endocytosis of a virion into the lumen of endosomes, the M2 ion channel is thought to cause acidification of the virion interior. In addition, the influenza virus M2 ion channel is thought to function in the exocytic pathway by equilibrating the pH gradient between the acidic lumen of the trans-Golgi network and the neutral cytoplasm. A necessary test of the proposed roles of the influenza virus M2 ion channel in the virus life cycle is to show directly that the M2 ion channel conducts protons. We have measured the ionic selectivity and activation of three subtypes (Udorn, Weybridge, and Rostock) of the M2 ion channel in oocytes of Xenopus laevis by measurement of 1) the intracellular pH (pHin) of voltage-clamped oocytes, 2) the current-voltage relationship in solutions of various pH and ionic composition, and 3) the flux of 86Rb. We took advantage of the low pHin achieved during incubation in low pH medium to study the effects of low pHin on M2 activation. Oocytes expressing each of the three subtypes of the M2 protein a) underwent a slow acidification when incubated in medium of low pH (acidification was blocked by the M2 ion channel inhibitor, amantadine); b) had current-voltage relationships that shifted to more positive values and had greater conductance when the pHout was lowered (this relationship was modified when Na- was replaced by NH4+ or Li+); c) had an amantadine-sensitive influx of Rb+. The effects on the current-voltage relationship of reduced pHin were opposed to the increased conductance found with reduced pHout. We interpret these results to indicate that the M2 ion channel is capable of conducting H+ and that other ions may also be conducted. Moreover, the channel conductance is reduced by decreased pHin. These findings are consistent with the proposed roles of the M2 protein in the life cycle of influenza A virus.     

Identification of a Ca2+/H+ antiport in the plant chloroplast thylakoid membrane

To assess the availability of Ca2+ in the lumen of the thylakoid membrane that is required to support the assembly of the oxygen-evolving complex of photosystem II, we have investigated the mechanism of 45Ca2+ transport into the lumen of pea (Pisum sativum) thylakoid membranes using silicone-oil centrifugation. Trans-thylakoid Ca2+ transport is dependent on light or, in the dark, on exogenously added ATP. Both light and ATP hydrolysis are coupled to Ca2+ transport through the formation of a transthylakoid pH gradient. The H+-transporting ionophores nigericin/K+ and carbonyl cyanide 3-chlorophenylhydrazone inhibit the transport of Ca2+. Thylakoid membranes are capable of accumulating up to 30 nmol Ca2+ mg-1 chlorophyll from external concentrations of 15 μM over the course of a 15-min reaction. These results are consistent with the presence of an active Ca2+/H+ antiport in the thylakoid membrane. Ca2+ transport across the thylakoid membrane has significant implications for chloroplast and plant Ca2+ homeostasis. We propose a model of chloroplast Ca2+ regulation whereby the activity of the Ca2+/H+ antiporter facilitates the light-dependent uptake of Ca2+ by chloroplasts and reduces stromal Ca2+ levels.     

Photosynthetic apparatus in chilling-sensitive plants

The levels of both tightly and loosely bound Mn in chloroplasts from fresh, cold and dark stored as well as illuminated leaves of Lycopersicon esculentum Mill. were studied in relation to Hill reaction activity. The tightly bound Mn pool represents one third of the total Mn content in chloroplasts isolated from the fresh leaves, and its level does not change following cold storage and illumination of leaves. Upon cold and dark storage of leaves the loss from the chloroplasts of about 40%–50% of the total amount of Mn is accompanied by an almost complete inactivation of the Hill reactions, as studied with water as an electron donor, as well as by the appearance of an EPR signal characteristic of free Mn²⁺ ions. Following illumination of such leaves, the restroration of Hill reaction activity is accompanied by an increase in the total Mn content in chloroplasts of up to 70%–80% of the Mn level measured in the fresh leaves and by disappearance of the EPR signal. In contrast, aging in the cold of isolated chloroplasts does not affect their Mn content. The addition of manganese does not result in the restoration of Hill reaction activity in chloroplasts from cold stored leaves but causes a restoration of this activity inhibited by linolenic acid. The data suggest that the loosely bound Mn pool (extractable with Tris) can be differentiated into two fractions: (1) one functionally inactive in electron transport and (2) one responsible for restoration of Hill reaction activity. Mn of the latter fraction (about 45% of the total Mn content) probably originates from the free Mn ions present in the interior of the chloroplasts following the cold and dark storage of leaves and from Mn reincorporated into chloroplasts from the cytoplasm. Incorporation of Mn from both these sources into thylakoid membrane to form a functionally active, loosely bound Mn pool proceeds during the illumination of leaves and results in the restoration of Hill reaction activity inhibited following the storage of leaves in dark and cold.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - Diquat 1,1-ethylene 2,2-dipiridylium dibromide - EPR electron paramagnetic resonance - FFA free fatty acid - MV methyl viologen, N,N-dimethyl-4,4 dipyridyldihydrochloride - Tris tris-(hydroxymethyl) aminomethane     

Modulation of the 6-position of benzopyran derivatives and inhibitory effects on the insulin releasing process

The synthesis of different series of 4- and 6-substituted R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans is described. All of these new benzopyran derivatives were bearing, at the 4-position, a phenylthiourea moiety substituted on the phenyl ring by a meta or a para-electron-withdrawing group such as Cl or CN. The study aimed at exploring the influence of the nature of the substituent at the 6-position in order to develop new benzopyran-type K(ATP) channel activators exhibiting an improved selectivity towards the insulin secreting cells. The original compounds were examined in vitro on rat pancreatic islets (inhibition of insulin release) as well as on rat aorta rings (vasorelaxant effect) and their activity was compared to that of the reference K(ATP) channel activators (±)-cromakalim, (±)-pinacidil, diazoxide and to previously synthesized cromakalim analogues. Structure-activity relationships indicated that the inhibitory effect on the insulin secreting cells was related to the lipophilicity of the molecules and to the size of the substituent located at the 6-position. A marked inhibitory activity on the insulin secretory process was obtained with molecules bearing a bulky tert-butyloxycarbonylamino group at the 6-position (20-23). The latter compounds were found to have the same efficacy on the pancreatic endocrine tissue than some previously described molecules. Lastly, radioisotopic experiments further identified R/S-N-4-chlorophenyl-N'-(6-tert-butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (23) as a K(ATP) channel opener.