Damage of cell membranes by linoleic acid hydroperoxide

It has been shown on Ehrlich ascite carcinoma cells that under the effect of linoleic acid hydroperoxides in vitro ionic permeability and membrane capacity of the cells sharply decrease after some threshold concentration of hydroperoxides (greater than 10(-5) M), while the threshold value decreases with the increase of the time of cell incubation in the presence of hydroperoxides. Interrelationship between the development of induced POL processes in the cell membranes and disturbance of their functional-structural state in the living cell is discussed.     

gamma-Amino butyric-N-acid sensitivity of mouse and human oocytes

gamma-Aminobutyric acid (GABA)-sensitivity was studied in mouse and human oocytes using electrophysiological techniques. Isolated mouse oocytes at the germinal vesicle (GV) or metaphase II stage, and human oocytes at the GV stage or following resumption of meiosis in culture, were sensitive to GABA. The transmitter usually hyperpolarized the membrane, with a concomitant decrease followed by an increase in membrane conductance, at threshold concentrations as low as 10(-10) M. GABA response was not evoked in Cl-free medium. Bicuculline (10(-5)-10(-6) M) reversibly blocked GABA (10(-9)-10(-4) M) responses. In contrast mouse fertilized eggs were insensitive to GABA at concentrations of 10(-5) M or lower. A possible biological role of the neurotransmitter GABA is discussed.     

Myocardial Depolarizing Response to Glutamate in the Myogenic Heart of the Branchiopod Crustacean Triops longicaudatus

Fine structure of the heart and the effects on the heartbeat of some transmitter candidates in crustacean cardioregulatory system were examined in the myogenic heart of the branchiopod crustacean Triops longicaudatus. Electron microscopy revealed that, in each myocardial cell, myofibrils are confined in the part facing the epicardium and intercalated disks are present between the myofibrillar regions of adjacent myocardial cells. No neural elements were found in the heart, suggesting lack of extrinsic cardioregulatory nerves from the central nervous system. Gamma aminobutyric acid and acetylcholine produced no detect-able changes in the myogenic activity of the heart at concentrations up to 10(-3) M, respectively. Glutamate induced a depolarizing membrane response in the cardiac muscle with a threshold concentration of approximately 1x10(-5) M. The amplitude of the depolarizing response was concetration-dependent and saturated at approximately 1x10(-4) M. The myogenic activity of the heart increased in frequency with glutamate of less than approximately 3x10(-5) M. With higher dose of glutamate, action potential adaptation occurred in the cardiac muscle and the heart exhibited a systolic arrest.     

Potent constriction of cat coronary arteries by hydroperoxides of arachidonic acid and its blockade by anti-inflammatory agents

The omega-6 and omega-9 hydroperoxides of arachidonic acid caused dose-dependent constriction of cat coronary arteries in concentrations of 10(-8) to 10(-5) M. Their potency was comparable to that of prostaglandin (PG) E2, and PGF2 alpha and 100 times greater than that of arachidonic acid. The cyclooxygenase inhibitor, meclofenamate markedly reduced constriction caused by the hydroperoxides but potentiated constriction caused by the prostaglandins. The effects of the hydroperoxides were also reduced by indomethacin and dexamethasone but were unaffected by the thromboxane synthetase inhibitor imidazole. Since the hydroperoxides are not substrates for cyclooxygenase, it is suggested that they have a direct effect on the arteries which can be antagonized by anti-inflammatory drugs.     

Bioelectrorheological model of the cell. 5. Electrodestruction of cellular membrane in alternating electric field.

Recently proposed analysis of the extensil stress developed in a cellular membrane subjected to an alternating electric field (Pawłowski, P., and M. Fikus, 1993. Bioelectrorheological model of the cell. 4. Analysis of the extensil deformation of the membrane in an alternating field. Biophys. J. 65:535-540) was applied in calculations of extensil stress threshold values, sigma eo[d], producing experimentally observed electrodestruction of cells within the frequency range of 7 x 10(1) - 3 x 10(5) Hz. It was shown that the susceptibility (s[d] = 1/sigma eo[d]), of the membrane to this process varies with field frequency and depends on the type of cells. Electrodestruction is facilitated in the 10(5)-Hz field. A rheological hypothesis explaining the experimentally observed dependence of membrane stability on electric field frequency was proposed and successfully tested for two other phenomena: electroporation and electrofusion.     

Flow cytometric estimation of transmembrane potential of macrophages--a comparison with microelectrode measurements

Potential-dependent accumulation of the lipophilic cationic dye 3,3' dihexyloxacarbocyanine (DiOC6(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose-dependent (10(-5) M-10(-7) M) and time-dependent (0 min-45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential-dependent at dye concentrations between 5 X 10(-8) M and 10(-7) M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.     

A critical evaluation of the effect of sorbitol on the ferric-xylenol orange hydroperoxide assay

Measurement of hydroperoxide concentration by the ferric-xylenol orange assay has the advantages of simplicity, convenience, and inertness to oxygen (Gay, C., et al. (1999) Anal. Biochem. 273, 149-155). However, its sensitivity is limited by the molar absorption coefficients, which are less than 50,000 M(-1) cm(-1) for most hydroperoxides. An earlier report showed that this could be significantly enhanced by the inclusion of 100 mM sorbitol in the assay solution, resulting in an increase of the apparent absorption coefficient for H(2)O(2) from 4.46 x 10(4) to 2.24 x 10(5) M(-1) cm(-1) (Wolff, S. P. (1994) Methods Enzymol. 233, 182-189). It was claimed that the technique was also valid for other hydroperoxides, such as butyl and cumyl. In an attempt to extend this modification to a wider range of hydroperoxides, we have confirmed the enhancement of the assay of H2O2 by sorbitol. However, the sensitivity of the measurements of butyl, cumyl, amino acid, protein, and human blood serum hydroperoxides was only approximately doubled by the inclusion of sorbitol. A mechanism explaining the difference in the assay of H2O2 and the organic hydroperoxides is proposed.     

Effect of 3,5,3'-triiodo-L-thyronine on amino acid accumulation and membrane potential in Sertoli cells of the rat testis

The purpose of this study was to investigate the effect of T3 on amino acid accumulation and on the membrane potential of Sertoli cells of immature rat testes. Testes of pre-pubertal and pubertal rats were pre-incubated (30 min) in Krebs-Ringer bicarbonate buffer and incubated in the presence of [14C]methylaminoisobutyric acid with and without T3 for 15, 45 and 60 min. The hormone (10(-6) M and 10(-7) M) significantly stimulated amino acid accumulation in 6 and 13-day old rat testes but did not have any effect in neonatal and pubertal animals. T3 produced a dose-dependent hyperpolarizing effect at concentrations of 10(-6) M, 10(-5) M, 2 x 10(-5) M and 10(-4) M. We conclude that T3 induces a membrane hyperpolarization in Sertoli cells and stimulates amino acid accumulation in immature rat testes, demonstrating that the hormone has a rapid plasma membrane action.     

5-Hydroxytryptamine stimulates fluid secretion in locust malpighian tubules independently of cAMP

5-Hydroxytryptamine (5-HT) stimulates fluid secretion by semi-isolated Malpighian tubules of Locusta in a dose-dependent manner. The threshold of stimulation is between 10(-8) and 10(-7) M 5-HT; maximal activation occurs at doses greater than 10(-6) M. Relative to the activation induced by diuretic hormone (storage lobe extracts), 5-HT increases the rate of fluid secretion by only 65%. Phentolamine, the alpha-adrenergic blocker, failed to inhibit either DH or 5-HT stimulated secretion. Diuretic hormone raises the levels of intracellular of cAMP, and activates adenylate cyclase in plasma membrane preparations of Locusta Malpighian tubules. 5-HT (10(-4) M) has no effect in either assay system. Thus 5-HT can stimulate fluid secretion independently of cAMP. A hypothetical model for hormone stimulated fluid secretion by Locusta Malpighian tubules, involving dual-receptor activation, is proposed. Other biogenic amines, including octopamine, adrenalin, dopamine, synephrine and the formamidine chlordimeform were tested for their ability to stimulate fluid secretion. Only dopamine showed a weakly stimulatory effect.     

Neuromuscular transmission in an insect visceral muscle

The electrical properties of the muscles of locust oviduct have been examined using intracellular recordings. The muscle cells are both dye and electrically coupled. They possess a wide array of spontaneous electrical activity ranging from slow oscillations of membrane potential to action potentials. In addition to possessing spontaneous electrical activity, certain regions of the oviduct are under motor control. The amplitude of evoked excitatory junction potentials (EJPs) increased step wise revealing innervation from a maximum of three motor units. These EJPs underwent summation and facilitation, and reached a critical threshold at which point the membrane revealed an active response. Bath applied glutamate, aspartate, proctolin, and octopamine were tested for their ability to alter resting potential and EJPs. L-glutamate (1.6 X 10(-5) M and above) produced a dose-dependent depolarization of membrane potential accompanied by a reduction in amplitude of EJPs. Although L-aspartate resulted in similar effects, the concentrations required were higher than those for glutamate. Proctolin (6.3 X 10(-11) M-6.0 X 10(-9) M) resulted in a dose-dependent depolarization but had little or no effect on amplitude of EJPs. Application of D, L-octopamine (3.2 X 10(-5) M-1.7 X 10(-4) M) induced a small hyperpolarization and a reduction in amplitude of EJP. It is suggested that contractions of locust oviduct appear to be regulated by a combination of a classical neurotransmitter such as glutamate, along with the neuromodulators octopamine and proctolin.     

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution

Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides has been performed, as a model for the reactions of lipid hydroperoxides with vitamin E radical in biological systems. The rates of reaction of hydroperoxides (n-butyl hydroperoxide 1, sec-butyl hydroperoxide 2, and tert-butyl hydroperoxide 3) with vitamin E radical (5,7-diisopropyl-tocopheroxyl 4) in benzene solution have been determined spectrophotometrically. The second-order rate constants, k-1, obtained are 1.34 x 10(-1) M-1s-1 for 1, 2.42 x 10(-1) M-1s-1 for 2, and 3.65 x 10(-1) M-1s-1 for 3 at 25.0 degrees C. The result indicates that the rate constants increase as the total electron donating capacity of the alkyl substituents at alpha-carbon atom of hydroperoxides increases. The above rates, k-1, are about seven order of magnitude lower than those, k1, for the reaction of vitamin E with peroxyl radical.     

GRF is a highly potent activator of adenylate cyclase in the normal human, bovine and rat pituitary: interaction with somatostatin

The effect of GRF adenylate cyclase activation was studied in normal human, bovine and rat pituitary tissues. Human GRF (hGRF) activates adenylate cyclase in normal human pituitary membrane preparations in a concentration dependent manner (ED5 0 = 10(-11) M). In bovine pituitary cells hGRF stimulates GH secretion into the medium (ED5 0 = 7 X 10(-12) M) and activates adenylate cyclase (ED5 0 = 10(-11) M). In normal rat pituitary cells in monolayer culture, rat GRF (rGRF) stimulates adenylate cyclase (ED5 0 = 3 X 10(-11) M). In normal human pituitary membrane preparations and in normal rat pituitary cells in culture, somatostatin inhibits GRF-stimulated adenylate cyclase in a non-competitive manner, while it does not affect basal (i.e. non-stimulated) adenylate cyclase levels. VIP, a peptide which is structurally homologous to hGRF and rGRF is a weak GRF-agonist and activates adenylate cyclase in human and rat pituitary preparations at concentrations greater than 10 nM.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of serotonin and leu-enkephalin on the habituating central neurons of Helix pomatia L. in situ and in vitro

1. Habituating neurons (a, b and c) of Helix pomatia reacted to the serotonin (10(-5)-10(-4)M) with depolarization evoking oscillatory waves and burst firing at the range of -35 to -55 MP values. 2. Isolated habituating cells were hyperpolarized by serotonin and failed to generate membrane oscillation or bursting pattern. 3. Only the isolated habituating neurons reacted to the application of leu-enkephalin (10(-5)-10(-4)M) by depolarization. 4. Neither membrane oscillation nor burst firing were evoked by leu-enkephalin. 5. On the cells a, b and c leu-enkephalin modulated the serotonin effect through cyclic 3',5'-AMP system both in situ and in vitro. 6. The membrane oscillation and burst firing of the habituating cells are connected to the regulation of various rhythmic processes including pneumostoma movements.     

Respiratory resistance of methylotrophic bacteria to formate and cyanide

Whole cells and cell-free preparations of the methylotrophic bacteria, Pseudomonas sp. AM 1 and Achromobacter parvulus, can oxidize formate at tis concentration in the reaction medium up to 1 M. The respiration of whole cells is registered at a concentration of formate greater than 10(-2) M, while that of cell-free extracts at a formate concentration greater than 5 X 10(-5) M. This seems to be due to the presence of a permeability barrier in cells for formate. The oxidation of reduced TMPD and exogenous cytochrome c by the membrane preparations of the two bacteria is inhibited by formate and cyanide; Ki50% = 2.5 X 10(-2) and 10(-6) M, respectively. The oxidation of NADH by the membrane preparations of the bacteria is not inhibited by 1 M formate and 5 X 10(-4) M cyanide but is inhibited by formaldehyde with Ki50% = 3 X 10(-2) M. Formaldehyde has no effect on the oxidation of reduced TMPD and cytochrome c at concentrations greater than 2 X 10(-1) M. These data indicate that respiration of the studied methylotrophic bacteria in the presence of high formate concentrations should be attributed in the presence of a branched electron transport chain in them; one branch of the chain is resistant to formate and cyanide, but is sensitive to formaldehyde.     

Interactions between estrogen, tamoxifen, octylphenol, and two polychlorinated biphenyls in murine splenocytes.

Prior exposure of cultured murine splenocytes to 17beta-estradiol (E) protects them from the membrane disrupting effects of the xenoestrogen 4-tert-octylphenol (OP). Using splenocytes isolated from male Balb/c mice, we tested whether (a) the xenoestrogen, 2', 3', 4', 5'-tetrachloro-4-biphenylol (PCB-OH), or the polychlorinated biphenyl, 3, 3', 4, 4'-tetrachlorobiphenyl (PCB 77), which displays both estrogenic and anti-estrogenic actions, would compromise the membrane integrity of the cells and (b) E or tamoxifen (TX), another ligand for the E receptor, would protect the membranes of cells exposed to the agents. We also examined possible interactions between OP, PCB-OH, and PCB 77 on the cells. Splenocytes were cultured for 24 hr. Concentrations of OP (10(-5)-10(-9) M), PCB-OH (10(-6)-10(-16) M), or PCB 77 (10(-8)-10(-12) M) significantly compromised the membrane integrity of the cultured splenocytes in a dose response manner. Concentrations of E as high as 10(-5) M or TX as high as 10(-7) M were without effect. Incubation of splenocytes in medium containing E or TX at 10(-7) M for 2 hr prior to the subsequent addition of either OP, PCB-OH or PCB 77 (final concentrations of 10(-7), 10(-7), or 10(-8) M, respectively) blocked the membrane disrupting effects. Incubation of splenocytes in medium containing 10(-7) M E starting 2 hr after the addition of OP or PCB 77 or incubation of splenocytes in medium containing 10(-7) M TX starting 2 hr after the addition of OP or PCB-OH did not block the damaging effects of OP, PCB 77, or PCB-OH on the cell membranes. No interactions were observed when various combinations of OP, PCB-OH, or PCB 77 were used. These data suggest that: (a) TX acts like E in this system, (b) a prior response of splenocytes to E or TX can protect them from the potential cytotoxic effects of OP, PCB-OH, or PCB 77; and, (c) OP, PCB-OH, and PCB 77 were not additive in their actions.     

Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: effects of inhibitors of cyclic AMP phosphodiesterase and Na+-K+-ATPase.

Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10(-3) M) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7 X 10(-6) M), Persantin (5 X 10(-5) M) or RO-20-1724 (10(-4) M), prevents cell replication induced by PMA or serum. In contrast, ouabain (10(-4) M) and N,N'-dicyclohexylcarbodiimide (10(-5) M), inhibitors of Na+-K+-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G1 phase at the time of reinitiation of the cell cycle from a stationary (Go) phase, a second is associated with the G1-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane enzymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner.     

Plasma membrane potential modulates chemotactic peptide-stimulated cytosolic free Ca2+ changes in human neutrophils

The relationship between fMet-Leu-Phe-induced changes in the cytosolic free Ca2+ concentration [( Ca2+]i), plasma membrane potential depolarization, and metabolic responses was studied in human neutrophils. Receptor-activated depolarization occurred both at high and resting [Ca2+]i, but was inhibited at very low [Ca2+]i. Phorbol 12-myristate 13-acetate-induced plasma membrane depolarization, on the contrary, was independent of [Ca2+]i. The threshold fMet-Leu-Phe concentration for plasma membrane depolarization (10(-8) M) was at least 1 log unit higher than that for [Ca2+]i increases (5 X 10(-10) M) and coincident with that for NADPH oxidase activation. Nearly maximal [Ca2+]i increases were elicited by 3 X 10(-9) fMet-Leu-Phe in the absence of any significant plasma membrane potential change. This observation allowed us to investigate the effects of artificially induced plasma membrane depolarization and hyperpolarization at low fMet-Leu-Phe concentrations (10(-9) to 3 X 10(-9) M) which did not perturb plasma membrane potential. Depolarizing (gramicidin D at 10(-7) to 10(-6) M or KCl at 50 mM) and hyperpolarizing (valinomycin at 4 microM) treatments had little influence on unstimulated [Ca2+]i levels, whereas fMet-Leu-Phe-induced transients were significantly altered. Gramicidin D and KCl decreased the fMet-Leu-Phe-induced [Ca2+]i increases in Ca2+-containing or in Ca2+-free media. Valinomycin, on the contrary, increased receptor-stimulated [Ca2+]i increases, and the effect was larger in the presence of extracellular Ca2+. Valinomycin also strongly potentiated secretion. It is suggested that plasma membrane depolarization in human neutrophils is a physiological feedback mechanism inhibiting receptor-dependent [Ca2+]i changes.     

The effect of ouabain on the sugar receptor of the contact chemoreceptive sensilla of the fly Protophormia terraenovae]

Studies have been made on the inhibitory effect of ouabain solutions on bioelectrical activity of the labellar sensillae of flied. It was shown that 10(-2) M ouabain solution irreversibly inhibits the activity, where as 10(-3) and 10(-4) M concentrations decrease the frequency of impulses within 40-60 min. Ouabain solution is a specific stimulator of the sugar receptor of the sensillae with a threshold of 10(-7) M; in combination with 0.2 M glucose, it inhibits impulse activity with the first 200 msec of stimulation. The effect is observed in a narrow zone of ouabain concentrations, with a maximum approximately at 10(-4) M. Differences between the effects of the inhibitor at the vicinity of the onset of generator potential and those in the impulse activity zones on the membrane of the sensory cell are suggested.     

Effects of histamine on contractile activity of lymphatic node capsules. The NO role

Effects of histamine (10(-9)--5 x 10(-5) M) on the phase and tonic contractile activity of capsular smooth muscles of isolated bovine mesentery lymph node were investigated. Dual dose-depended effect of histamine was found. Low concentrations of histamine less than 10(-7) M caused a decrease of contractile activity, whereas higher concentrations of histamine (more than 5 x 10(-7) M) resulted in increase of the phase and tonic contractions. Both H1- and H2-receptors of smooth muscle cells are involved in the response. Much of the relaxing histamine-induced response is produced by the stimulation of the endothelial cells. We believe that activating effect of histamine is due to the excitation of H1-receptors located on the membrane of myocytes, whereas its inhibitory effect occurs in two ways: 1) via excitation of H2-receptors located on the membrane of myocytes; 2) via stimulation of the NO production by the endothelial cells of lymph node sinus.