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.     

Participation of ACTH1–10 and ACTH4–10 on the melatonin modulation of benzodiazepine receptors in rat cerebral cortex

In this study, we examined the effect of intracerebroventricular (i.c.v) injection of melatonin and/or ACTH_1–10 and ACTH_4–10 on [³H]flunitrazepam binding sites in the cerebral cortex of hypophysectomized rats. Hypophysectomy increased the B_max (maximum number of binding sites) of benzodiazepine (BNZ) receptors for at least 7 days after surgery, without changing K_D (dissociation constant). The i.c.v. injection of melatonin to hypophysectomized rats significantly increased B_max, whereas the same doses of melatonin were ineffective in sham-operated animals. In both cases, K_D values were unchanged. The i.c.v injection of ACTH_1–10 to hypophysectomized animals significantly increased B_max, an effect that was enhanced by simultaneous i.c.v. injection of ACTH_1–10+melatonin, reaching higher values of B_max than the i.c.v. injection of these hormones individually. No significant changes in K_D values were found after ACTH_1–10 and/or melatonin administration. However, the i.c.v. injection of ACTH_4–10 to hypophysectomized rats did not change B_max, although it significantly increased K_D values, indicating a decrease in the BNZ binding affinity. Melatonin injection counteracted this effect of ACTH_4–10, returning K_D to the control value. Moreover, although the lower dose of i.c.v. melatonin used, 10 ng, was unable to modify B_max of BNZ binding in the ACTH_4–10-injected group, the higher dose, 20 ng, significantly increased B_max. The results suggest that these ACTH-derived peptides can modulate the effect of melatonin on brain benzodiazepine receptors.     

Nature of the FeO2 bonding in myoglobin: An overview from physical to clinical biochemistry

Summary The iron(II)-dioxygen bond in myoglobin and hemoglobin is a subject of wide interest. Studieas range from examinations of physical-chemical properties dependent on electronic structure, to investigations of stability as a function of oxygen supply. Stability properties are of particular importance in vivo, since the oxygenated form is known to be oxidized easily to the ferric form, which cannot be oxygenated and is therefore physiologically inactive.Kinetic and thermodynamic studies of the stability of native oxymyoglobin have revealed a new feature in FeO₂ bonding. In vivo. the iron center is always subject to a nucleophilic attack of the water molecule or hydroxyl ion, which can enter the heme pocket from the surrounding solvent, and thereby irreversibly displace the bound dioxygen from MbO₂ in the form of O ₂ ^– so that the iron is converted to the ferric form. A free energy diagram for the potential reactions of FeO₂ visualizes myoglobin as a molecular structure that can provide in solution the delicate balance of kinetic and thermodynamic factors necessary to stabilize reversible oxygenation, as opposed to irreversible autoxidation to metmyoglobin.     

One lipid, multiple functions: how various pools of PI(4,5)P2 are created in the plasma membrane

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a minor lipid of the inner leaflet of the plasma membrane that controls the activity of numerous proteins and serves as a source of second messengers. This multifunctionality of PI(4,5)P₂ relies on mechanisms ensuring transient appearance of PI(4,5)P₂ clusters in the plasma membrane. One such mechanism involves phosphorylation of PI(4)P to PI(4,5)P₂ by the type I phosphatidylinositol-4-phosphate 5-kinases (PIP5KI) at discrete membrane locations coupled with PI(4)P delivery/synthesis at the plasma membrane. Simultaneously, both PI(4)P and PI(4,5)P₂ participate in anchoring PIP5KI at the plasma membrane via electrostatic bonds. PIP5KI isoforms are also selectively recruited and activated at the plasma membrane by Rac1, talin, or AP-2 to generate PI(4,5)P₂ in ruffles and lamellipodia, focal contacts, and clathrin-coated pits. In addition, PI(4,5)P₂ can accumulate at sphingolipid/cholesterol-based rafts following activation of distinct membrane receptors or be sequestered in a reversible manner due to electrostatic constrains posed by proteins like MARCKS.     

Is there a critical tissue oxygen tension for bioenergetic status and cellular pH regulation in solid tumors?

Bioenergetic and metabolic status have been correlated with tissue oxygenation in murine fibrosarcomas (FSaII) of varying sizes (44–600 mm³). Ratios of -nucleoside triphosphates to inorganic phosphate (NTP/P_i) and phosphocreatine to inorganic phosphate (PCr/P_i) ratios derived from³¹P nuclear magnetic resonance spectroscopy (NMR) were positively correlated to median tissue O₂ tension (pO₂) values using O₂-sensitive needle electrodes. pH declined during growth with intracellular acidosis being evident in tumors >350 mm³. Whereas lactic acid formation greatly contributed to this decline in small and medium-sized tumors, adenosine triphosphate (ATP) hydrolysis and slowing down of the activities of pumps involved in cellular pH regulation seem to be major factors responsible for intracellular acidification in bulky tumors. PCr levels decreased at an early growth stage, whilst ATP concentrations dropped in bulky malignancies only, coinciding with a decrease in adenylate energy charge and a substantial rise in the levels of total P_i On average, median pO₂ values of ca. 10 mmHg represent a critical threshold for energy metabolism. At higher median O₂ tensions, levels of ATP, phosphomonoester (PME) and total P_i were relatively constant. This coincided with intracellular alkalosis or neutrality and stable adenylate ratios. On average, median pO₂ values <10 mmHg coincided with intracellular acidosis, ATP depletion, a drop in energy charge and rising P_i levels.     

The chloride effect in human hemoglobin: A new kind of allosteric mechanism

In human hemoglobin hydrogen ions, chloride, 2,3-diphosphoglycerate and CO₂ cooperate to shift the oxygen equilibrium curve to the right. Bovine hemoglobin, by contrast, has an intrinsically low oxygen affinity: when stripped, it is as low as that of human hemoglobin in the presence of 0.1 M NACl+0.1 M DPG.     

Shortening velocity of single muscle cells isolated from a molluscan smooth muscle

Summary The maximal unloaded shortening velocity (V_max) of smooth muscle cells isolated from the pedal retractor muscle ofMytilus was more than twice as large as that of the whole muscle, suggesting the presence of extracellular components which resist the contraction of the whole muscle. The V_max of the isolated cells was almost constant at cell lengths ranging between 0.5 and 0.831₀ (1₀, optimal length for tension generation) indicating that the intracellular resistance to contraction is negligible within this range of lengths.     

Amino acid absorption in jejunum and ileum in vivo — a kinetic comparison of function on surface area and regional bases

Summary Maximum absorptive capacities (V_max) for the jejunum and ileum corrected for the presence of an unstirred layer of water have been calculated for glycine, valine and methioninein vivo in fowls per unit surface area and per region. V_max per cm² showed that ileal enterocytes had a greater absorbing capacity than jejunal for glycine and valine but not for methionine. V_max for glycine and valine, calculated for the whole jejunum and ileum, however, were not different but for methionine the jejunal value was 1.9 times greater than the ileal.MAM is supported by an A.R.C. research grant (No. A50/37).     

Substance P-induced fibrinolysis in the forearm of healthy humans

Physiological saline with or without substance P (50 ng/ml) was infused into the humeral artery in 6 healthy males. Indices of fibrinolytic activity (whole blood diluted lysis time, euglobulin lysis time, lysis areas in non-heated fibrin plates produced by plasma or euglobulin precipitate, plasminogen plasma levels, ₂-macroglobulin, Cl-inhibitor, and ₂-antiplasmin) were evaluated in the homolateral antecubital vein before and after 5 min of substance P or saline infusion. After substance P the fibrinolytic activity increased, as can be seen from the shortening of lysis times (p<0.01) and enlargement of the lysis areas (p<0.01). A reduction of plasminogen plasma levels (p<0.01), associated with a decrease in ₂-anitplasmin (p<0.01), was also found. Alpha₂-macroglobulin and Cl-inhibitor were instead unaltered by the peptide. The saline infusion, on the other hand, was unable to modify any of the examined indices. We concluded that exogenous substance P given intra-arterially increases fibrinolytic activity in locally-sampled venous blood through a mechanism which remains to be elucidated.     

On partial and differential bleaching experiments with the visual pigments in a fresh water euryhaline teleost (Etroplus suratensis)

Summary Visual pigment was extracted from a fresh water toleost,Etroplus suratensis and the optical density measured over a range of 300–650 nm. The absorption spectrum indicates the _max at 550 nm and a small hump at 530 nm. Through partial bleaching at 630 nm, it was confirmed that the fish possesses a mixture of 2 visual pigments: the one with the _max at 550 nm predominating over the other.     

Hypotensive activity of histidine-containing analogues of C-terminal hexapeptide of Substance P

Summary Four new hexapeptide analogues of C-terminal Substance P fragment with increased solubility in aqueous solutions are described. The peptides contain histidine in positions 6, 8, 9 and 10, respectively. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of amino acid residues in various positions in the C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive and antigenic properties, respectively. Only the [His⁶] SP_6-11 analogue had an unchanged antigenic structure when compared with the C-terminal region of Substance P, but it showed an almost total loss of hypotensive activity. The [His⁹] SP_6-11 analogue retained 50% of the hypotensive activity of the C-terminal hexapeptide but showed a markedly reduced expression of the antigenic epitope localized in this region of Substance P.     

Sphingosine 1-phosphate increases glucose uptake through trans-activation of insulin receptor

Sphingosine 1-phosphate (S1P) is a bioactive lipid that acts through a family of G-protein-coupled receptors. Herein, we report evidence of a novel redox-based cross-talk between S1P and insulin signaling pathways. In skeletal muscle cells S1P, through engagement of its S1P₂ receptor, is found to produce a transient burst of reactive oxygen species through a calcium-dependent activation of the small GTPase Rac1. S1P-induced redox-signaling is sensed by protein tyrosine phosphatase-1B, the main negative regulator of insulin receptor phosphorylation, which undergoes oxidation and enzymatic inhibition. This redox-based inhibition of the phosphatase provokes a ligand-independent trans-phosphorylation of insulin receptor and a strong increase in glucose uptake. Our results propose a new role of S1P, recognizing the lipid as an insulin-mimetic cue and pointing at reactive oxygen species as critical regulators of the cross-talk between S1P and insulin pathways. Any possible implication of S1P-directed insulin signaling in diabetes and insulin resistance remains to be established.     

The effect of ligand on the kinetics of the reaction of the sulfhydryl groups of human hemoglobin with p-mercuribenzoate

Summary The rate of reaction of PMB with the masked sulfhydryls of human hemoglobin derivatives correlates well with the extent of dissociation for various ligands. It is suggested that the ₁₂ dimer of hemoglobin participates in the slow step of the reaction with PMB.     

Effects of isoxazolyl-naphthoquinoneimines on growth and oxygen radical production inTrypanosoma cruzi andCrithidia fasciculata

Several 4-(aminomethylisoxazolyl)-1,2-naphthoquinones inhibited growth and DNA synthesis inTrypanosoma cruzi and stimulated O₂ uptake and generation by the parasite epimastigotes and their mitochondrial and microsomal membranes; these results support the idea that oxygen radicals play a role in quinone toxicity. Maximal effects on respiration and generation were observed with antimycin-inhibited cells. Similar results as well as stimulation of H₂O₂ production were obtained withCrithidia fasciculata despite the presence of catalase in this organism.Acknowledgments. This work was aided by grants from the University of Buenos Aires, the Scientific Office of the American States Organization and CEDIQUIFA (Buenos Aires). S.G.G. and M.P.M.P. are Research Fellows and A.O.M.S. is Career Investigator of CONICET (Argentina). L. T. Viñas and M. G. Gutierrez lent able technical assistance.     

Rotifer populations in plankton communities: Energetics and life history strategies

Rotifers play an important role in many freshwater plankton communities. The populations are controlled from bottom-up depending on different food quantities and qualities. As threshold food levels for rotifers are higher than for cladocerans they are often outcompeted when food concentrations are lowered by the clearance activity of cladocerans. Rotifers also are controlled from top-down by predators, especially by copepods, by instars ofChaoborus and by predatory rotifers. Mechanical interference by daphnids is considered here as a special case of predation. Different defense mechanisms are discussed. At the cost of higher food concentrations (high K_s-food levels) rotifers may exhibit high maximum growth rates (r_max) and short times for their population development. This ability increases with rotifer body size. Within this taxonomic unity, therefore, different life history strategies have developed. These strategies may be characterized by the r_max/K_s-model presented.     

Influence of phosphorus supply and light intensity on mycorrhizal response inPisum-Rhizobium-Glomus symbiosis

The influence of mycorrhizal colonization withGlomus mosseae on parameters of N₂ fixation and plant growth was studied in pot experiments with pea plants (Pisum sativum L.) infected withRhizobium leguminosarum and supplied with varied levels of phosphorus (P) and nitrogen (N). Reduced light intensities were used to evaluate the dependence of the microsymbionts on assimilate supply. In plants grown with low P supply, mycorrhization increased the concentration of P in shoots, and thus N₂ fixation. Reduced light intensity significantly depressed mycorrhizal colonization and nodule growth in low-P plants. When P supply did not limit plant growth and N₂ fixation, however, the percentage of mycorrhizal colonization was reduced due to the higher P status, and the microsymbionts were not impaired by low light intensities. To maximize carbohydrate supply, another experiment was carried out at high light intensity of 900 mol m^–2s^–1 and with non-limiting P supply. Nitrogen fertilization, given as starter N, enhanced plant growth, but delayed nodule formation. Towards flowering, nodulation rapidly increased, but less so inGlomus inoculated plants. After 28 days mycorrhizal plants were lower in shoot dry weight, nodule dry weight and nitrogenase activity. The results suggest that under many, but not all, environmental conditions the host plant is able to restrict mycorrhizal colonization and, thus, to prevent impairment ofRhizobium symbiosis.deceased in May 1994     

Influence of environmental conditions on the amount of N2O released from activated sludge in a domestic waste water treatment plant

Waste water purification is characterized by intensive mineralization and nitrification processes. Because of the high O₂ demand, temporarily anaerobic conditions may be produced, and denitrification by nitrifying organisms as well as heterotropic denitrification may contribute to N₂O release. In situ measurements (1993–1994) suggest that N₂O is released from activated sludge in a domestic waste water treatment plant at an average rate of 1040 g m^–2h^–1 with a range between zero and 6198 g m^–2h^–1. The production of N₂O seems to be related to the concentration of NO ₂ ^– and NO ₃ ^– as well as to the pH. In the waste water about 75–200 g N₂O l^–1 is dissolved. This N₂O is released after discharge into the receiving waters. The N₂O is produced essentially by nitrification rather than by heterotropic denitrification. On a long-term scale the increasing use of mechanical-biological waste water purification plants world-wide may add increasingly to the anthropogenic production of N₂O, although the present amount of N₂O produced is negligible compared to its global terrestrial production.     

Identification of tyrosine-phosphorylated proteins of the mitochondrial oxidative phosphorylation machinery

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H₂O₂, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase α chain is probably tyrosine-phosphorylated, but demonstrated that the β chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.Received 7 January 2005; received after revision 19 April 2005; accepted 22 April 2005     

Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate acetate stimulated human leukocytes

Summary A Ginkgo biloba extract (Gbe) containing flavonoids, among other compounds, was tested for the release of activated oxygen species ( , H₂O₂, OH^.) during the stimulation of human neutrophils (PMNs) by a soluble agonist. The extract slows down O₂-consumption (respiratory burst) of stimulated cells by its inhibitory action on NADPH-oxidase, the enzyme responsible for the reduction of O₂ to . Consequently, superoxide anion ( ) and hydrogen peroxide (H₂O₂) production is significantly decreased when the PMNs stimulation is done in the presence of the extract at concentrations of 500, 250 and 125 g/ml. Moreover, the hydroxyl radical generation (OH^.) is very much decreased at concentrations as low as 15.6 g Gbe/ml, which indicates that the extract also has free radical scavenging activity. Gbe is able at least to reduce very severel the activity of myeloperoxidase contained in neutrophils. This enzyme, secreted into the intra and extracellular medium, catalyzes the oxidation of chloride (Cl^–) by H₂O₂ to yield strong oxidants (HOCl, chloramines) which are implicated in inflammatory processes     

Role of adenosine A1 and A2 receptors in the regulation of aldosterone production in rat adrenal glands

Summary To investigate the roles of adenosine A₁ and A₂ receptors in the regulation of aldosterone production, we examined the effects of adenosine and adenosine agonists (N⁶-cyclohexyl adenosine; selective adenosine A₁ receptor agonist and 5-N-ethylcarboxamine adenosine; selective adenosine A₂ receptor agonist) on aldosterone and cyclic AMP production in rat adrenal capsular cells. Neither adenosine nor 5-N-ethylcarboxamine adenosine caused significant effects on basal aldosterone or cyclic AMP production. Also, adenosine (10^–3M) showed no consistent effects on aldosterone and cyclic AMP production induced by ACTH. On the other hand, N⁶-cyclohexyl adenosine exhibited a significant inhibition of basal aldosterone and cyclic AMP production at doses of 10^–4 M and 10^–3 M; furthermore, 10^–3 M N⁶-cyclohexyl adenosine inhibited aldosterone and cyclic AMP production stimulated by ACTH. These results suggest that adenosine A₁ receptors are coupled to and inhibit adenylate cyclase and may be involved in the inhibition of aldosterone production.