Changes of Plasma Membrane Properties in a Human Pre-T Cell Line Undergoing Apoptosis

A variety of cellular functions are modulated by the physical properties of the cell membrane, and the modification of intracellular transfer, resulting from loss of membrane integrity, may contribute toward setting the cell onto the pathway of apoptosis. Apoptosis in lymphoid cells can be induced in different ways and biochemical modifications occur at an early phase of cell death, while the morphological features of apoptosis are evident later. We previously reported that DMSO is an efficient apoptosis-inducing factor in the human RPMI-8402 pre-T cell line. The aim of the present study was to verify the effect of DMSO on the plasma membrane fluidity, the intracellular calcium concentration and the phosphodiesterase activity in DMSO-induced apoptosis. Our results show a modification of membrane fluidity associated with an increase of intracellular Ca²⁺ concentration. Moreover, we demonstrate that these modifications are related to a decrease in the phosphodiesterase (PDE) activity. The correlation between the proceedings of added DMSO and the induction of apoptosis will provide significant information regarding the first part of the apoptotic process.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

DNA damage induced by bleomycin in the presence of dibucaine is not predictive of cell growth inhibition

Growth inhibition and cell killing by bleomycin are believed to be related to the ability of this antibiotic to cleave chromosomal DNA. Because bleomycin has an intracellular site of action, its ability to cross biological membranes must be critical to its overall effectiveness as an antitumor agent. The local anesthetic dibucaine acts to enhance membrane fluidity; therefore, the reported ability of this local anesthetic to modulate bleomycin effects on KB cells was investigated. Cells were treated with various bleomycin congeners in the presence or absence of dibucaine for 24 h. Dibucaine enhanced the inhibition of cell growth mediated by bleomycin A2, demethylbleomycin A2, bleomycin B2, and isobleomycin A2. N-Acetylbleomycin A2 did not inhibit cell growth in the absence of dibucaine, but it was inhibitory in the presence of dibucaine. Cells treated simultaneously for analysis of DNA breakage on alkaline sucrose gradients revealed that breakage was also enhanced in the presence of dibucaine. The degree of enhancement varied with dose and bleomycin congener. N-Acetylbleomycin A2 did not induce DNA breakage in either the absence or the presence of dibucaine. While growth inhibition and net DNA breakage correlated reasonably well in the absence of dibucaine for each bleomycin analogue tested, proportionality was lost in the presence of dibucaine, and very little DNA breakage was present when growth inhibition was complete. These observations imply that, at least in the presence of dibucaine, bleomycin may mediate growth inhibition at some locus in addition to chromosomal DNA and, also, that a given net amount of bleomycin analogue induced DNA damage per se does not produce a specific degree of growth inhibition.     

Alterations in some membrane properties in rat brain following exposure to lead

The effect of lead exposure on intracellular calcium levels, membrane fluidity, lipid peroxidation, acetylcholinesterase and monoamine oxidase activity and its accumulation in different regions of the brain were studied to understand the molecular mechanism of lead induced neurotoxicity. Lead treatment (20 mg/kg lead nitrate, intraperitoneally, once daily for 15 days) resulted in a significant accumulation of lead in all brain regions with the maximum being in the hippocampus. Levels of glutathione, lipid peroxidation, intracellular calcium and membrane fluidity, as well as the activity of the membrane bound enzymes, acetylcholinesterase and monoamine oxidase, increased to a significant level in certain areas of the rat brain. The results suggest that lead exerts neurotoxic effects by altering certain membrane bound enzymes and may cause oxidative stress.     

Cyclosporine A Induces Apoptotic and Autophagic Cell Death in Rat Pituitary GH3 Cells

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels.     

3–Nitropropionic Acid–Induced Changes in Bilayer Fluidity in Synaptosomal Membranes: Implications for Huntington's Disease

The use of 3–nitropropionic acid (3–NP) and other mitochondria inhibitors to effectuate animal models of Huntington's disease has been well established. 3–NP administration has been shown to lead to pathology similar to that of HD, including massive loss of striatal neurons associated with oxidative stress. Oxidative stress induced by 3–NP also extends to the cortex, an area where little neuron loss occurs. No mechanism as of yet accounts for selective loss of striatal neurons while sparing cortical neurons. In the present study, a nitroxide stearate lipid bilayer-specific spin-label was utilized to probe 3–NP-induced fluidity changes in striatal and cortical synaptosomal membranes. In cortical synaptosomes, membrane fluidity increased in animals previously treated with 3–NP when compared to controls injected with saline vehicle, while in striatal synaptosomes, membrane fluidity decreased in animals treated with 3–NP when compared to controls. The results of the present study suggest that oxidatively-induced changes in membrane fluidity may be involved in mechanisms by which selective striatal neuronal loss occurs in this animal model of Huntington's disease.     

Investigation of the TEA-resistant outward current in the somatic membrane of perfused nerve cells

Outward currents remaining after addition of 20–50 mM of tetraethylammonium (TEA) ions to the extracellular or intracellular solution, were investigated in perfused isolatedHelix neurons. After this addition, the inactivated inward current carried by potassium ions, the potential-dependent and kinetic characteristics of which differ from those of potassium outward currents suppressed by TEA, is preserved in the membrane. A component dependent on the inward calcium current was found in this TEA-resistant outward current; it was abolished by replacement of the extra-cellular calcium ions by magnesium ions, by blocking of the calcium channels by extracellular cadmium ions, and by their destruction by intracellular fluoride ions. Increasing the intracellular concentration of free calcium ions by perfusing the cell with solutions containing calcium-EGTA buffer potentiated the TEA-resistant component of the outward current, whereas removal of these ions with EGTA weakened it. It is concluded that a system of outward current channels whose activation depends on the presence of calcium ions near the inner surface of the membrane is present in the somatic membrane. It is suggested that to keep these channels capable of being activated, calcium ions must bind with the structures forming their internal opening.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 460–468, September–October, 1979.     

Mechanisms of cell death of thymocytes induced by polyunsaturated, monounsaturated and trans-fatty acids

Polyunsaturated fatty acids (PUFAs) are rapidly cytotoxic to isolated murine thymocytes, and the degree of cell death has been correlated with changes in membrane fluidity, elevation of intracellular calcium concentration and generation of reactive oxygen species. We have compared the degree of cell death and increase in membrane fluidity of C-20 and C-22 omega-3 and 6 PUFAs to those induced by monounsaturated and trans-fatty acids, and find that concentrations which induce comparable increases in membrane fluidity do not cause comparable cell death. The C-18 omega-6 causes a decrease in membrane fluidity, yet is the most potent in causing cell death. Omega-6 PUFAs are more cytotoxic than omega-3 PUFAs, while monounsaturated and trans-fats show little cytotoxicity and only at much higher concentrations. Cell death is preceded by reductions of both plasma and mitochondrial membrane potential, and occurs via apoptosis. These results indicate that cell death is due to mechanisms other than changes in membrane fluidity.     

Protective Effect of Sucrose and Sodium Chloride for Lactococcus lactis during Sublethal and Lethal High-Pressure Treatments

The bactericidal effect of hydrostatic pressure is reduced when bacteria are suspended in media with high osmolarity. To elucidate mechanisms responsible for the baroprotective effect of ionic and nonionic solutes, Lactococcus lactis was treated with pressures ranging from 200 to 600 MPa in a low-osmolarity buffer or with buffer containing 0.5 M sucrose or 4 M NaCl. Pressure-treated cells were characterized in order to determine viability, the transmembrane difference in pH (ΔpH), and multiple-drug-resistance (MDR) transport activity. Furthermore, pressure effects on the intracellular pH and the fluidity of the membrane were determined during pressure treatment. In the presence of external sucrose and NaCl, high intracellular levels of sucrose and lactose, respectively, were accumulated by L. lactis; 4 M NaCl and, to a lesser extent, 0.5 M sucrose provided protection against pressure-induced cell death. The transmembrane ΔpH was reversibly dissipated during pressure treatment in any buffer system. Sucrose but not NaCl prevented the irreversible inactivation of enzymes involved in pH homeostasis and MDR transport activity. In the presence 0.5 M sucrose or 4 M NaCl, the fluidity of the cytoplasmic membrane was maintained even at low temperatures and high pressure. These results indicate that disaccharides protect microorganisms against pressure-induced inactivation of vital cellular components. The protective effect of ionic solutes relies on the intracellular accumulation of compatible solutes as a response to the osmotic stress. Thus, ionic solutes provide only asymmetric protection, and baroprotection with ionic solutes requires higher concentrations of the osmolytes than of disaccharides.     

The respiration of isolated rat hepatic cells in suspension

1. Rat-hepatic cells in suspension have been shown to have an endogenous respiration of 5·6±0·17 when suspended in 0·1 m-sucrose and 0·02 m-tris–hydrochloric acid buffer. The respiration in 0·25 m-sucrose and 0·02 m-tris–hydrochloric acid buffer is 30–40% less. 2. Potassium chloride (0·05 m) is slightly inhibitory and calcium chloride (0·0025 m) highly inhibitory to endogenous respiration of the hepatic cells in suspension. The cells do not respire in Krebs–Ringer phosphate buffer. 3. The respiration of the hepatic cells in suspension is stimulated by pyruvate, citrate, isocitrate, oxoglutarate, succinate, fumarate, malate and glutamate; there is no significant stimulation (or inhibition) by glucose, fructose, acetate and butyrate. In almost all the cases where stimulation was observed, it was found that the higher the endogenous respiration the lower is the stimulation.     

Membrane fluidity of microsomal and thymocyte membranes after X-ray and UV irradiation

A brief literature review shows that ionizing radiation in biological membranes and in pure lipid membranes causes malondialdehyde formation, indicating lipid peroxidation processes. With respect to membrane fluidization by ionizing radiation, in pure lipid membranes rigidization effects are always reported, whereas contradictory results exist for biological membranes. Starting from the assumption that membrane proteins at least partly compensate for radiation effects leading to a rigidization of membrane lipid regions, pig liver microsomes, as a representative protein-rich intracellular membrane system, were irradiated with X-rays or UV-C with doses up to 120 Gy at a dose rate of 0.67 Gy min^–1 and up to 0.73 J cm^–2 at an exposure rate of 16.2 mJ cm^–2 min^–1, respectively. For both irradiation types a weak but significant positive correlation between malondialdehyde formation and membrane fluidity is revealed throughout the applied dose ranges. We conclude that the membraneous protein lipid interface increases its fluidity under radiation conditions. Also, thymocyte ghosts showed an increased fluidity after X-ray irradiation. Fluidity measurements were performed by the pyrene excimer method.     

Cyclosporin A Rescues Thymocytes from Apoptosis Induced by Very Low Concentrations of Thapsigargin: Effects on Mitochondrial Function

Raising intracellular calcium levels can induce apoptosis or programmed cell death in many cells. While early rises in intracellular calcium are not universally associated with apoptotic cell death, calcium clearly plays a key role in many of the biochemical events which occur during apoptosis. In this paper we have determined intracellular calcium rises induced by 2, 10, and 100 nMthapsigargin in mouse thymocytes. These concentrations cause increases in cytosolic calcium of 100–250, 400–600, and >1000 nM,respectively. These rises are sustained for at least 85 min and the ratio between the maximum rise caused by 10 nMcompared to 2 nMthapsigargin is 2.1 ± 0.4 (n= 6). Both 2 and 10 nMthapsigargin cause apoptosis at 24 h as shown by DNA fragmentation and morphology when examined by electron microscopy. Cyclosporin A (CsA) inhibits apoptosis caused by 2 nMthapsigargin but not that caused by 10 nMthapsigargin. Electron microscopy of thymocytes treated with 2 nMthapsigargin at 24 h shows intact mitochondria although with altered morphology. There is no loss of ATP or decrease in the ATP/ADP ratio in these cells over 12 h. Mitochondria in cells treated with 10 nMthapsigargin, however, are swollen by 6 h and many are lost by 24 h. These cells show greatly diminished ATP content by 12 h and a decrease in ATP/ADP ratio. Examination of the effects of PMA, an activator of the plasma membrane calcium ATPase pump, on cells treated with 10 nMthapsigargin suggests that two pools of calcium may be responsible for the differential effects of the two calcium levels in the cells. Probing of the mitochondrial membrane potential (MMP) by rhodamine 123 staining of live cells shows that the collapse of the MMP caused by 10 nMthapsigargin is unaffected by CsA. The MMP is also reduced in cells treated with 2 nMthapsigargin but this is restored by CsA. Cells are also rescued from apoptosis caused by 2 nMthapsigargin by incubation with FK506. This immunosuppressive agent has no effect on the membrane permeability transition induced in isolated mitochondria. These results suggest that very low rises in intracellular calcium in thymocytes cause activation-induced cell death inhibited by CsA and FK506 and are without effect on ATP levels and therefore do not involve irreversible mitochondrial damage. Exceeding these calcium levels by only twofold results in apoptosis accompanied by reduced ATP levels and mitochondrial damage, although apoptotic cell death in this instance is unaffected by the classic inhibitor of mitochondrial permeability transition, CsA.     

Alterations in Membrane Lipid Dynamics of Leukemic Cells Undergoing Growth Arrest and Differentiation: Dependency on the Inducing Agent

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane–water–lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D₃and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-γ (IFN-γ) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-γ treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-γ resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.     

Effects of local anesthetics on membrane properties I. Changes in the fluidity of phospholipid bilayers

The effect of the local anesthetic dibucaine on the solid to liquid-crystalline phase transition in phospholipid vesicles was studied by calorimetry and fluorescence polarization. The partition coefficient (> 3000) of dibucaine in the membranes of vesicles prepared from acidic phospholipids was more than 20 times higher than in neutral phospholipid membranes under the same conditions. Calorimetric measurements on vesicles prepared form acidic phospholipids (bovine brain phosphatidylserine; dipalmitoylphosphatidylglycerol) showed that dibucaine (1 · 10⁻⁴M) produced a significant reduction in the gel-liquid crystalline transition temperature (T_c). This fluidizing effect of dibucaine on acidic phospholipid membranes was even more marked in the presence of Ca²⁺. In contrast, dibucaine at the same concentration did not alter the T_c of neutral phospholipids (dipalmitoylphosphatidylcholine). Significant increase in the fluidity of neutral phospholipid membranes occurred only at higher dibucaine concentrations (2 · 10⁻³M. Measurements of the fluorescence polarization and lifetime of the probe, 1,6-diphenylhexatriene, in acidic phospholipid vesicles revealed that dibucaine (1 · 10⁻⁴M caused an increase in the probe rotation rate indicating an increase in the fluidity of the phospholipid membranes. A good correlation was obtained between fluorescence polarization data on dibucaine-induced changes in membrane fluidity and calorimetric measurements on vesicles of the same type.     

Intracellular Calcium Changes in Neuronal Cells Induced by Alzheimer's ß-Amyloid Protein Are Blocked by Estradiol and Cholesterol

1. The elevation of intracellular Ca²⁺ levels ([Ca²⁺]_i) in immortalized hypothalamic neurons (GT1–7 cells) after exposure to Alzheimer's ß-amyloid protein (AßP[25–35]) was investigated using a multisite fluorometry system.2. The marked rise in [Ca²⁺]_i appeared afterexposure to 5–20-M AßP[25–35]. Analysis of the spatiotemporal patterns of [Ca²⁺]_i changes revealed that the magnitude and the latency of the response to AßP in each cell werehighly heterogeneous.3. The preadministration of 17ß-estradiol, 17-estradiol, phloretin and cholesterol, which influence the properties of membranes, such as membrane fluidity or membrane potential, significantly decreased the rise in [Ca²⁺]_i.4. These findings support the idea that disruption of calcium homeostasis by AßP channels may be the molecular basis of the neurotoxicity of AßP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.     

Cyclic GMP regulation of a voltage-activated K channel in dissociated Enterocytes

Summary Enterrocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from –23 to – 39 mV and 29 to 13 M, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at –80 mV and subsequently stepped to a series of depolarizing voltages (from–70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above –20 mV. This current was sensitive to holding potential and was not activated when the cells were held at –40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the currentcarrying ion. BaCl₂ (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC₅₀ for charybdotoxin was 50 nM. Cyclic. GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above–40 mV. Previous studies with intact flounder epithelium showed that K recycling and net K secretion were inhibited by Ba²⁺ and by cGMP. We suggest that the channel responsible for the whole-cell current described in this study may be important in K recycling and secretion.     

Ethanol inhibits muscarinic receptor-stimulated phosphoinositide metabolism and calcium mobilization in rat primary cortical cultures

In recent years, it has been hypothesized that muscarinic receptor-stimulated phosphoinositide (PI) metabolism may represent a relevant target for the developmental neurotoxicity of ethanol. Age-, brain region-, and receptor-specific inhibitory effects of ethanol on this system have been found, both in vitro and after in vivo administration. As a direct consequence of this action, alterations of calcium homeostasis would be expected, through alterations of inositol trisphosphate formation, which mediates intracellular calcium mobilization. In the present study, the effects of ethanol (50–500 mM) on carbachol-stimulated PI metabolism and free intracellular calcium levels were investigated in rat primary cortical cultures, by measuring release of inositol phosphates and utilizing the two calcium probes fluo-3 and indo-1 on an ACAS (Adherent Cell Analysis and Sorting) Laser Cytometer. Ethanol exerted a concentration-dependent inhibition of carbachol-stimulated PI metabolism. In addition, ethanol's inhibitory effect paralleled the temporal development of the muscarinic receptor signal transduction system, with the strongest inhibition (25–50%) occurring when maximal stimulation by carbachol occurs (days 5–7). Ethanol also exerted a concentration-dependent decrease in free intracellular calcium levels following carbachol stimulation. Both initial calcium spike amplitude, seen in all responsive cells, as well as the total number of cells responding to carbachol, were decreased by ethanol. The inhibitory effects of ethanol seemed dependent upon preincubation time, in that a longer preincubation (30 min) with the lowest dose (50 mM), showed almost the same decrease in responding cell number and reduction in spike amplitude in responding cells, as a shorter incubation (10 min) with the highest ethanol dose (500 mM). The specificity of the response to carbachol was demonstrated by blocking the response with 10 M atropine. Moreover, experiments with carbachol in calcium-free buffer with 1 mM EGTA indicated that the initial calcium spike was due to intracellular calcium mobilization from intracellular stores. Since calcium is believed to play important roles in cell proliferation and differentiation, these results support the hypothesis that this intracellular signal-transduction pathway may be a target for ethanol, contributing to its developmental neurotoxicity.     

Role of Pinoline and Melatonin in Stabilizing Hepatic Microsomal Membranes against Oxidative Stress

We investigated the influence of pinoline (0.01–1.5 mM) on microsomal membrane fluiditybefore and after rigidity was induced by oxidative stress. In addition, we tested the effect ofpinoline in the presence of 1 mM melatonin. The fluidity in rat hepatic microsomes wasmonitored using fluorescence spectroscopy and it was compared to the inhibition ofmalonaldehyde (MDA) plus 4-hydroxyalkenals (4-HDA) production as a reflection of lipid peroxidation.Below 0.6 mM, pinoline inhibited membrane rigidity in a manner parallel to its inhibitoryeffect on MDA + 4–HDA formation. At concentrations between 1–1.5 mM, pinoline wasless effective in stabilizing microsomal membranes than was predicted from its inhibition oflipid peroxidation. The addition of 1 mM melatonin enhanced the membrane-stabilizing activityof pinoline (0.01–0.6 mM). This cooperative effect was not observed for concentrations ofpinoline between 1–1.5 mM. When pinoline was tested without induced oxidative damage,1–1.5 mM pinoline maintained membrane fluidity at the same level as that recorded afterinduced lipid peroxidation. The results suggest that pinoline may be another pineal moleculethat prevents membrane rigidity mediated by lipid peroxidation and this ability is enhancedby melatonin.     

Cryopreservation of dog semen: the effects of Equex STM paste on plasma membrane fluidity and the control of intracellular free calcium

Understanding cryoinjury of dog spermatozoa is crucial to preserving fertilizing ability. This study examined flow cytometric indicators of sperm function to explore the reported benefits of Equex STM paste. The motility of cryopreserved spermatozoa immediately and 1h after thawing was higher in the extender containing 0.5% Equex; no significant differences between the two extenders were observed regarding viability, acrosomal integrity and intracellular Ca(2+) concentration. The proportion of spermatozoa having high membrane fluidity increased significantly post-thawing. The interaction between time after thawing and treatment was significant for plasma membrane fluidity. Dilution in a commercial diluent for transport before processing caused a significant increase in intracellular Ca(2+), which may affect functional survival. No significant difference with or without Equex was detected in plasma membrane fluidity. However, a significant interaction between Equex and dogs was detected. A significant decrease in intracellular Ca(2+) was detected in the live cell population both after dilution in Andersen's buffer and again after cooling and equilibration. One hour post-thaw, the proportion of live spermatozoa with high calcium concentration increased to a similar proportion as that seen in diluted semen; the interaction between diluent and dog was significant. The results suggest that Equex in the diluent benefited motility after cryopreservation. Live spermatozoa with high intracellular Ca(2+) after cryopreservation seem to have a favoured survival in the first hour after thawing. Nevertheless, survival after cryopreservation was severely compromised, explaining the relatively poor fertility of cryopreserved dog semen.     

Cardiotoxicity of calmidazolium chloride is attributed to calcium aggravation, oxidative and nitrosative stress, and apoptosis

The intracellular calcium concentration ([Ca]_i) regulates cell viability and contractility in myocardial cells. Elevation of the [Ca]_i level occurs by entry of calcium ions (Ca²⁺) through voltage-dependent Ca²⁺ channels in the plasma membrane and release of Ca²⁺ from the sarcoplasmic reticulum. Calmidazolium chloride (CMZ), a subgroup II calmodulin antagonist, blocks L-type calcium channels as well as voltage-dependent Na⁺ and K⁺ channel currents. This study elaborates on the events that contribute to the cytotoxic effects of CMZ on the heart. We hypothesized that apoptotic cell death occurs in the cardiac cells through calcium accumulation, production of reactive oxygen species, and the cytochrome c-mediated PARP activation pathway. CMZ significantly increased the production of superoxide (O₂^•–) and nitric oxide (NO) as detected by FACS and confocal microscopy. CMZ induced mitochondrial damage by increasing the levels of intracellular calcium, lowering the mitochondrial membrane potential, and thereby inducing cytochrome c release. Apoptotic cell death was observed in H9c2 cells exposed to 25 μM CMZ for 24 h. This is the first report that elaborates on the mechanism of CMZ-induced cardiotoxicity. CMZ causes apoptosis by decreasing mitochondrial activity and contractility indices and increasing oxidative and nitrosative stress, ultimately leading to cell death via an intrinsic apoptotic pathway.