Attenuation in gap junctional coupling has consistently been associated with induction of rapid or synchronous cell division in normal and pathological conditions. In the case of the v-src oncogene, gating of Cx43 gap junction channels has been linked to both direct phosphorylation of tyrosines (Y247 and 265) and phosphorylation of serine target of Erk1/2 (S255, 279, and 282) on the cytoplasmic C-terminal domain of Cx43. However, only the latter has been associated with acute rather than chronic gating of the channels immediately after v-src expression, a process that is mediated through a “ball and chain” type mechanism. In this study we show that while ERK1/2 is necessary for acute closure of gap junction channels, it is not sufficient. Rather, multiple pathways converge to regulate Cx43 coupling in response to expression of v-src, including parallel signaling through PKC and MEK1/2, with additional positive and negative regulatory effects mediated by PI3kinase, distinguished by the involvement of Akt.
Cx43; Gap Junctions; v-Src; Erk1/2; signal transduction
We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to “regular,” round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10–20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane–cytoskeleton interaction and cell motility.
Electroporation; Nanosecond pulsed electric field; Colloid-osmotic swelling; Blebbing; Membrane protrusion; Cell motility
The objective of this investigation was to evaluate postmortem changes of electric charge of human erythrocytes and thrombocytes after fatal carbon monoxide (CO) poisoning. The surface charge density values were determined on the basis of the electrophoretic mobility measurements of the cells carried out at various pH values of electrolyte solution. The surface charge of erythrocyte membranes after fatal CO poisoning as well as after sudden unexpected death increased compared to the control group in the whole range of experimental pH values. Also, a slight shift of the isoelectric point of erythrocyte membranes to high pH values was observed. The surface charge of thrombocyte membranes after fatal CO poisoning decreased at low pH compared to the control group. However, at high pH, the values increased compared to the control group. The isoelectric point of thrombocyte membranes after fatal CO poisoning was considerably shifted toward low pH values compared to the control group. The observed changes are probably connected with the destruction of blood cell structure.
Fatal carbon monoxide poisoning; Surface charge; pH measurement; Erythrocyte; Thrombocyte
Connexins form channels with large aqueous pores that mediate fluxes of inorganic ions and biological signaling molecules. Studies aimed at identifying the connexin pore now include a crystal structure that provides details of putative pore-lining residues that need to be verified using independent biophysical approaches. Here we extended our initial cysteine-scanning studies of the TM1/E1 region of Cx46 hemichannels to include TM2 and TM3 transmembrane segments. No evidence of reactivity was observed in either TM2 or TM3 probed with small or large thiol-modifying reagents. Several identified pore residues in E1 of Cx46 have been verified in different Cx isoforms. Use of variety of thiol reagents indicates that the connexin hemichannel pore is large and flexible enough, at least in the extracellular part of the pore funnel, to accommodate uncommonly large side chains. We also find that that gating characteristics are largely determined by the same domains that constitute the pore. These data indicate that biophysical and structural studies are converging towards a view that the N-terminal half of the Cx protein contains the principal components of the pore and gating elements, with NT, TM1 and E1 forming the pore funnel.
channel pore; voltage gating connexin; cysteine scanning; chimera; hemichannels
Gap junctions (GJs) are composed of tens to many thousands of double-membrane spanning GJ channels that cluster together to form densely packed channel arrays (termed GJ plaques) in apposing plasma membranes of neighboring cells. In addition to providing direct intercellular communication (GJIC, their hallmark function), GJs, based on their characteristic double-membrane-spanning configuration, likely also significantly contribute to physical cell-to-cell adhesion. Clearly, modulation (up-/down-regulation) of GJIC and of physical cell-to-cell adhesion is as vitally important as the basic ability of GJ formation itself. Others and we have previously described that GJs can be removed from the plasma membrane via the internalization of entire GJ plaques (or portions thereof) in a cellular process that resembles clathrin-mediated endocytosis. GJ endocytosis results in the formation of double-membrane vesicles (termed annular gap junctions [AGJs] or connexosomes) in the cytoplasm of one of the coupled cells. Four recent independent studies, consistent with earlier ultrastructural analyses, demonstrate the degradation of endocytosed AGJ vesicles via autophagy. However, in TPA-treated cells others report degradation of AGJs via the endo-/lysosomal degradation pathway. Here we summarize evidence that supports the concept that autophagy serves as the cellular default pathway for the degradation of internalized GJs. Furthermore, we highlight and discuss structural criteria that seem required for an alternate degradation via the endo-/lysosomal pathway.
Autophagy; Cell-Cell Junctions; Connexin 43; Gap Junctions; Protein Degradation; Ubiquitin
There is abundant evidence showing that connexins form gap junctions. Yet, this does not exclude the possibility that connexins can exert other functions, separate from that of gap junction (or even a permeable hemichannel) formation. Here, we focus on these “non-canonical” functions of Connexin43 (Cx43), particularly in the heart. We describe two specific examples: The importance of Cx43 on intercellular adhesion, and the role of Cx43 in the function of the sodium channel. We propose that these two functions of Cx43 have important repercussions on the propagation of electrical activity in the heart, irrespective of the presence of permeable gap junction channels. Overall, the gap junction-independent functions of Cx43 in cardiac electrophysiology emerge as an exciting area of future research.
Connexin43; heart; arrhythmias; cell-cell adhesion
Oculodentodigital dysplasia (ODDD) is a rare developmental disease resulting from germline mutations in the GJA1 gene that encodes the gap junction protein connexin43 (Cx43). In addition to the classical ODDD symptoms that affect the eyes, teeth, bone and digits, in some cases ODDD patients have reported bladder impairments. Thus, we chose to characterize the bladder in mutant mouse models of ODDD that harbor two distinct Cx43 mutations, G60S and I130T. Histological assessment revealed no difference in bladder detrusor wall thickness in mutant compared to littermate control mice. The overall localization of Cx43 in the lamina propria and detrusor also appeared to be similar in the bladders of mutant mice with the exception that the G60S mice had more instances of intra-cellular Cx43. However, both mutant mouse lines exhibited a significant reduction in the phosphorylated P1 and P2 iso-forms of Cx43, while only the I130T mice exhibited a reduction in total Cx43 levels. Interestingly, Cx26 levels and distribution were not altered in mutant mice as it was localized to intracellular compartments and restricted to the basal cell layers of the urothelium. Our studies suggest that these two distinct genetically modified mouse models of ODDD probably mimic patients who lack bladder defects or other factors, such as aging or co-morbidities, are necessary to reveal a bladder phenotype.
Connexin; Cx43; Mutant; Bladder; Mutant mouse; Gap junction
Osteoblasts sense and respond to mechanical stimuli in a process involving influx and release of large ions and signaling molecules. Unapposed gap junction hemichannels formed of connexin43 (Cx43) have been proposed as a major route for such exchange, and in particular for release of ATP and prostaglandin E2 (PGE2) in osteocytes. However, we have found that Cx43-null osteoblasts have unaltered mechanically-induced PGE2 release and ATP-induced YoPro dye uptake. In contrast, PGE2 release in response to fluid shear stress is abolished in P2X7 receptor (P2X7R) null osteoblasts and ATP-induced dye uptake is attenuated following treatment of wildtype cells with a P2X7R or Pannexin1 (Panx1) channel blocker. These data indicate that Panx1 channels, in concert with P2X7R, likely form a molecular complex that performs the hemichannel function in osteoblast mechanosignaling.
P2X7R; ATP; osteoblast; gap junction; dye uptake
Although a functional pore domain is required for connexin 37 (Cx37)–mediated suppression of rat insulinoma (Rin) cell proliferation, it is unknown whether functional hemichannels would be sufficient or if Cx37 gap junction channels are required for growth suppression. To test this possibility, we targeted extracellular loop cysteines for mutation, expecting that the mutated protein would retain hemichannel, but not gap junction channel, functionality. Cysteines at positions 61 and 65 in the first extracellular loop of Cx37 were mutated to alanine and the mutant protein (Cx37-C61,65A) expressed in Rin cells. Although the resulting iRin37-C61,65A cells expressed the mutant protein comparably to Cx37 wild-type (Cx37-WT)–expressing Rin cells (iRin37), Cx37-C61,65A expression did not suppress the proliferation of Rin cells. As expected, iRin37-C61,65A cells did not form functional gap junction channels. However, functional hemichannels also could not be detected in iRin37-C61,65A cells by either dye uptake or electrophysiological approaches. Thus, failure of Cx37-C61,65A to suppress the proliferation of Rin cells is consistent with previous data demonstrating the importance of channel functionality to Cx37’s growth-suppressive function. Moreover, failure of the Cx37-C61,65A hemichannel to function, even in low external calcium, emphasizes the importance of extracellular loop cysteines not only in hemichannel docking but also in determining the ability of the hemichannel to adopt a closed configuration that can open in response to triggers, such as low external calcium, effective at opening Cx37-WT hemichannels.
Gap junction; Connexin; Hemichannel; Growth suppression; Insulinoma; Cx37
Glutamate scanning mutagenesis was used to assess the role of the calcicludine binding segment in regulating channel permeation and gating using both Ca2+ and Ba2+ as charge carriers. As expected, wild-type L-type calcium channels had a Ba2+ conductance ~2× that in Ca2+ (GBa/GCa = 2) and activation was ~10 mV more positive in Ca2+ vs. Ba2+. Of the eleven mutants tested, F1126E was the only one mutant that showed unique permeation and gating properties compared to wild-type. F1126E equalized the L-channel conductance (GBa/GCa = 1) and activation voltage-dependence between Ca2+ and Ba2+. Ba2+ permeation was reduced because the interactions among multiple Ba2+ ions and the pore were specifically altered for F1126E, which resulted in Ca2+-like ionic conductance and unitary current. However, high affinity block of monovalent cation flux was not altered for either Ca+2 or Ba2+. The half activation voltage of F1126E in Ba2+ was depolarized to match that in Ca2+, which was unchanged from wild-type. As a result, the voltages for half activation and half inactivation of F1126E in Ba2+ and Ca+2 were similar to those of wild-type in Ca+2. This effect was specific to F1126E since F1126A did not affect the half activation voltage in either Ca+2 or Ba2+. These results indicate that residues in the outer vestibule of the L-channel pore are major determinants of channel gating, selectivity and permeation.
mutation; voltage-dependent; surface charge; outer vestibule; selectivity filter; pore; electrophysiology
Connexin43 (Cx43) forms 22 s gap junctions that couple the granulosa cells of ovarian follicles. In Cx43 knockout mice, follicle growth is restricted due to impaired granulosa cell proliferation. We have used these mice to examine the importance of specific Cx43 phosphorylation sites in follicle growth. Serines at residues 255, 262, 279 and 282 are MAP kinase substrates that, when phosphorylated, reduce junctional conductance. Mutant forms of Cx43 were constructed with these serines replaced with amino acids that cannot be phosphorylated. These mutants were transduced into Cx43 knockout ovarian somatic cells which were combined with wildtype oocytes and grafted into immunocompromised female mice permitting follicle growth in vivo. Despite residues 255 or 262 being mutated to prevent their being phosphorylated, recombinant ovaries constructed with these mutants were able to rescue the null phenotype, restoring complete folliculogenesis. In contrast, Cx43 with serine to alanine mutations at both residues 279 and 282 or at all four residues failed to rescue folliculogenesis; the mutant molecules were largely confined to intracellular sites, with few gap junctions. Using an in vitro proliferation assay, we confirmed a decrease in proliferation of granulosa cells expressing the double mutant construct. These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development.
Cx43; phosphorylation; folliculogenesis; oocyte growth; granulosa cell; proliferation
Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 µM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro- 2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43- expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.
Connexins; Electrophysiology; Gap junction; Gap junctions; Gap junctions/cell–cell channels
Gap junctions and gap junction communication have long been recognized to play roles in tissue organization and remodeling through both cell autonomous and intercellular means. We hypothesized that these processes become dysregulated during pancreas cancer progression. Molecular and histological characterization of the gap junction protein, connexin43, during progression of pancreatic ductal adenocarcinoma could yield insight into how these events may contribute to or be modulated during carcinogenesis. In a mouse model of pancreatic ductal adenocarcinoma generated through targeted endogenous expression of KrasG12D in the murine pancreas, we examined the evolving expression and localization of connex-in43. Overall, connexin43 expression increased over time, and its localization became more widespread. At early stages, connexin43 is found almost exclusively in association with the basolateral membrane of duct cells found in invasive lesions. Connexin43 became increasingly associated with the surrounding stroma over time. Connexin43 phosphorylation was also altered during tumorigenesis, as assessed by migrational changes of the protein in immunoblots. These data suggest a potential role for gap junctions and connexin43 in mediating interactions between and amongst the stromal and epithelial cells in pancreatic ductal adenocarcinoma.
Connexin; Gap junction; Pancreas cancer; Phosphorylation
A great deal is now known about the protein components of tight junctions and adherens junctions, as well as how these are assembled. Less is known about the molecular framework of gap junctions, but these also have membrane specializations and are subject to regulation of their assembly and turnover. Thus, it is reasonable to consider that these three types of junctions may share macromolecular commonalities. Indeed, the tight junction scaffolding protein zonula occluden-1 (ZO-1) is also present at adherens and gap junctions, including neuronal gap junctions. On the basis of these earlier observations, we more recently found that two additional proteins, AF6 and MUPP1, known to be associated with ZO-1 at tight and adherens junctions, are also components of neuronal gap junctions in rodent brain and directly interact with connexin36 (Cx36) that forms these junctions. Here, we show by immunofluorescence labeling that the cytoskeletal-associated protein cingulin, commonly found at tight junctions, is also localized at neuronal gap junctions throughout the central nervous system. In consideration of known functions related to ZO-1, AF6, MUPP1, and cingulin, our results provide a context in which to examine functional relationships between these proteins at Cx36- containing electrical synapses in brain—specifically, how they may contribute to regulation of transmission at these synapses, and how they may govern gap junction channel assembly and/or disassembly.
AF6; Cingulin; Connexin36; Electrical synapse; Gap junctions; MUPP1; Neurons; PDZ domain
Gap junction channels composed of connexins connect cells, allowing intercellular communication. Their cellular assembly involves a unique quality-control pathway. Some connexins [including connexin43 (Cx43) and Cx46] oligomerize in the trans-Golgi network following export of stabilized monomers from the endoplasmic reticulum (ER). In contrast, other connexins (e.g., Cx32) oligomerize early in the secretory pathway. Amino acids near the cytoplasmic aspect of the third transmembrane domain have previously been shown to determine this difference in assembly sites. Here, we characterized the oligomerization of two connexins expressed prominently in the vasculature, Cx37 and Cx40, using constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) or treatment with brefeldin A to block ER vesicle trafficking. Both methods led to intracellular retention of connexins, since the cells lacked gap junction plaques. Retention of Cx40 in the ER prevented it from oligomerizing, comparable to Cx43. By contrast, ER-retained Cx37 was partially oligomerized. Replacement of two amino acids near the third transmembrane domain of Cx43 (L152 and R153) with the corresponding amino acids from Cx37 (M152 and G153) resulted in early oligomerization in the ER. Thus, residues that allow Cx37 to oligomerize early in the secretory pathway could restrict its interactions with coexpressed Cx40 or Cx43 by favoring homomeric oligomerization, providing a structural basis for cells to produce gap junction channels with different connexin composition.
Endothelium; Membrane traffic; Quality control; Chaperone; ERp29
In cardiomyocytes, β1-adrenergic receptor (β1-AR) plays an important role in regulating cardiac functions. Upon continuous ligand stimulation, β1-AR is internalized and mostly recycled back to the plasma membrane (PM). The recycling endosome (RE) is one of the membranous organelles involved in the protein recycling pathway. To determine whether RE is involved in the internalization of β1-AR upon ligand stimulation, we evaluated the localization of β1-AR after stimulation with a β-agonist, isoproterenol (Iso), in β1-AR-transfected COS-1 cells. After 30 min of Iso treatment and cell surface labeling with the appropriate antibodies, β1-AR was internalized from PM and translocated into the perinuclear region, the same location as the transferrin receptor, an RE marker. We then evaluated whether sorting nexin 27 (SNX27) participated in the β1-AR recycling pathway. When β1-AR and SNX27 were coexpressed, β1-AR coimmunoprecipitated with SNX27. In addition, shRNA-mediated silencing of SNX27 compromised β1-AR recycling and enhanced its delivery into lysosome. Overall, β1-AR on PM was internalized into RE upon Iso stimulation and recycled by RE through binding with SNX27 in COS-1 cells.
β1-Adrenergic receptor; Desensitization; Recycling endosome; SNX27
This study was conducted with the purpose of researching the effect of lycopene application on lipoprotein, paraoxonase (PON) and cytokines that are projected to be used in the diagnosis and treatment of diabetes by making experimental diabetes. At the end of a 1-month trial period, under ether anesthesia with jelly tubes, blood samples were taken from rat hearts. Blood samples were centrifuged and serum was obtained. From the serum samples, HbA1c, paraoxonase activity, lipoprotein levels and cytokines were determined. HbA1c levels and PON activity were found to be p < 0.001. At the triglyceride level, with regard to the control group, in all the groups a significant rise occurred (p ≤ 0.001). At the cholesterol level, with regard to the control group, a decline was observed in the other groups (p < 0.05). At the VLDL level, with regard to the control group, a significant rise was observed in the other groups (p < 0.05). At the HDL (p < 0.001) and LDL (p < 0.05) levels, with regard to the control group, a significant decline was observed in the other groups. At the TNF-α, IL-2, IL-6 and IL-10 levels no difference was found (p > 0.05). Experimental diabetes models have an important place in analyzing diabetes complications and determining treatment approaches.
Experimental diabetes mellitus; Rat; Lycopene; Lipoprotein; Paraoxonase; Cytokine
The aim of the study was to identify and determine the percent content of polyphenols in extracts from leaves and hawthorn bark, to examine the effect of the extracts on the properties of the biological membrane as well as to determine their antioxidant activity toward membrane lipids. In particular, a biophysical investigation was conducted on the effect of hawthorn extracts on the osmotic resistance and morphology of erythrocyte cells and on the packing of the heads of membrane lipids. Analysis of the polyphenol content of extracts used the HPLC method. Analysis of the polyphenol composition has shown a dominant share of procyanidins and epicatechin in both extracts. The research showed that the polyphenolic compounds contained in hawthorn extracts are incorporated mainly into the hydrophilic part of the erythrocyte membrane, inducing echinocyte shapes. They also diminish the packing order of the lipid polar heads of the membrane, as evidenced by the lowered generalized polarization values of Laurdan. The substances used induced increased osmotic pressure of erythrocytes, making them less sensitive to changes in osmotic pressure. The presence of the extract compounds in the outer hydrophilic part of the erythrocyte membrane, evidenced by examination of the shapes and packing in the hydrophilic part of membrane, indicates that the substances constitute a kind of barrier that protects the erythrocyte membrane against free radicals, while the membrane-bound extracts do not disturb the membrane structure and, thus, do not cause any side effects.
Hawthorn extract; Erythrocyte; Natural lipid; Antioxidant activity; Lipid organization; Osmotic resistance
It has been suggested that oxidative stress may play an important role in the pathogenesis of chronic otitis media (COM), but the role of oxidative stress in the pathogenesis of COM has not yet been fully explored. Therefore, the aim of this study was to investigate serum myeloperoxidase (MPO) activity, 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), total antioxidant capacity (TAC) and nitric oxide (NO) in patients with COM. Sixty-one patients with COM and 30 controls were enrolled in the present study. Patients were divided into two groups according to the presence (n = 21) or absence (n = 40) of cholesteatoma. Serum MPO activity and 4-HNE, MDA and NO levels were significantly higher in patients with COM than controls (for all, p < 0.001), while TAC levels were significantly lower (for all, p < 0.001). Serum MPO activity and MDA, 4-HNE and NO levels were significantly higher in patients with cholesteatoma than in those without cholesteatoma, while TAC levels were significantly lower; but the difference between groups was not statistically significant (p > 0.05). Increased oxidative stress seems to be associated with decreased antioxidant levels in patients with COM. Thus, increased oxidative stress may play a role in the pathogenesis of COM. It is believed that the administration of antioxidant vitamins such as A, C and E may be useful in preventing and treating COM.
Chronic otitis media; Cholesteatoma; Myeloperoxidase activity; 4-Hydroxynonenal; Malondialdehyde; Total antioxidant capacity; Nitric oxide
The mouse connexin 36 (Cx36) gene was mapped on chromosome 2 and an identical transcriptional start site was determined in brain and retina on exon I. Rabbit polyclonal antibodies to the presumptive cytoplasmic loop of the Cx36 protein recognized in immunohistochemical analyses Cx36 expression in the retina, olfactory bulb, hippocampus, inferior olive and cerebellum. In olivary neurons strong punctate labeling at dendritic cell contacts and weaker labeling in the cytoplasm of dendrites were shown by immuno electron microscopy. After expression of mouse Cx36 cDNA in human HeLa cells, neurobiotin transfer was increased 1.8-fold and electrical conductance at least 15-fold compared to untransfected HeLa cells. No Lucifer Yellow transfer was detected in either untransfected or Cx36 transfected HeLa cells. Single Cx36 channels in transfected HeLa cells showed a unitary conductance of 14.3 ± 0.8 pS. The sensitivity of Cx36 channels to transjunctional voltage was low in both HeLa-Cx36 cells and Xenopus oocytes expressing mouse Cx36. No increased transfer of neurobiotin was detected in heterotypic gap junctions formed by Cx36 and 9 other connexins expressed in HeLa cells. Our results suggest that Cx36 channels function as electrical synapses for transmission of electrical and metabolic signals between neurons in the central nervous system.
Gap junctions; Electrical synapses; Neuronal connexin; Transcriptional start site; Cx36
We investigated both the effect of levosimendan and the role of oxidant/antioxidant status and trace element levels in the pulmonary artery of rats. Fourteen male Wistar albino rats were randomly divided into two groups of seven animals each. Group 1 was not exposed to levosimendan and served as a control. Levosimendan (12 μg/kg) diluted in 10 ml 0.9 % NaCl was administered intraperitoneally to group 2. Animals of both groups were killed after 3 days, and their pulmonary arteries were harvested to determine changes in tissue oxidant/antioxidant status and trace element levels. The animals in both groups were killed 72 h after the levosimendan exposure treatment, and pulmonary arteries were harvested to determine levels of the lipid peroxidation product MDA and the antioxidant GSH as well as the decreased activity of antioxidant enzymes such as SOD, GSH-Px and CAT. It was found that MDA levels increased in pulmonary artery tissues of rats after levosimendan administration. The GSH level decreased in the pulmonary artery of rats after levosimendan treatment. Co, Mn, Fe, Cd and Pb levels were significantly higher (P < 0.001) and Mg, Zn and Cu levels significantly lower (P < 0.001) in the levosimendan group compared to the control group. These results suggest that levosimendan treatment caused an increase in free radical production and a decrease in antioxidant enzyme activity in the pulmonary artery of levosimendan-treated rats. It also caused a decrease or increase in the levels of many minerals in the pulmonary artery, which is an undesirable condition for normal pharmacological function.
Levosimendan; Oxidative stress; Free radical; Oxidant/antioxidant; Pulmonary artery
Alkylphosphocholines (APCs) belong to a class of synthetic antitumor lipids, which are new-generation anticancer agents. In contrast to traditional antitumor drugs, they do not attack the cell nucleus but, rather, the cellular membrane; however, their mechanism of action is not fully understood. This work compared the interactions of selected APCs [namely, hexadecylphosphocholine (miltefosine), octadecylphosphocholine and erucylphosphocholine] with the most important membrane lipids [cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] and examined their influence on a model membrane of tumor and normal cells. As a simple model of membranes, Langmuir monolayers prepared by mixing cholesterol either with a saturated phosphatidylcholine (DPPC), for a normal cell membrane, or with an unsaturated one (POPC), for a tumor cell membrane, have been applied. The APC–lipid interactions, based on experimental surface pressure (π) versus mean molecular area (A) isotherms, were analyzed qualitatively (with mean molecular area values) as well as quantitatively (with the ΔGexc function). Strong attractive interactions were observed for mixtures of APCs with cholesterol, contrary to the investigated phosphatidylcholines, for which the interactions were found to be weak with a tendency to separation of film components. In ternary monolayers it has been found that the investigated model systems (cholesterol/DPPC/APC vs cholesterol/POPC/APC) differ significantly as regards the interactions between film-forming molecules. The results demonstrate stronger interactions between the components of cholesterol/POPC/APC monolayers compared to cholesterol/POPC film, mimicking tumor cell membranes. In contrast, the interactions in cholesterol/DPPC/APC films were found to be weaker than those in the cholesterol/DPPC system, serving as a model of healthy cell membranes, thus proving that the incorporation of APCs is, from a thermodynamic point of view, unfavorable for binary cholesterol/DPPC monolayers. It can be concluded that the composition of healthy cell membranes is a natural barrier preventing the incorporation of APCs into normal cells.
Alkylphosphocholine; Model membrane; Langmuir monolayer; Interaction
The effect of monovalent ion (Li+, Na+, Cs+) interaction with monolayers of phosphatidylcholine (lecithin, PC) was investigated at the air/water interface. We present surface tension measurements of lipid monolayers obtained using a Langmuir method as a function of monovalent ion concentration. Measurements were carried out at 22 °C using a Teflon trough and a Nima 9000 tensiometer. Interactions between lecithin and monovalent ions result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of PC–monovalent ion complexes (PC−Me+).
Phosphatidylcholine; Monovalent ion; Complex formation equilibria; Monolayer; Langmuir trough
Phospholipids are ubiquitous in nature and are essential for the lipid bilayer of cell membranes. Their structural and functional properties are pivotal for the survival of the cell. In this study the phospholipids of healthy and cancerous human renal tissues from the same patients are compared with special reference to the electric charge of the membrane. A simple and highly effective normal-phase method is described for analyzing phospholipids content. This work is focused on changes of phospholipids content (PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; PtdEtn, phosphatidylethanoloamine; PtdCho, phosphatidylcholine) in cell membranes of renal cancer of pT1 stage, G2 grade, without metastasis. Surface charge density of healthy and cancerous human renal tissues was measured by electrophoresis. The measurements were carried out at various pH of solution. Depending on the surface charge density as a function of pH, acidic (CTA) and basic (CTB) functional group concentrations and their average association constants with hydrogen (KAH) or hydroxyl (KBOH) ions were evaluated. The process of cancer transformation was accompanied by an increase in total amount of phospholipids as well as an increase in CTA and KBOH, whereas KAH and CTB were decreased compared with unchanged tumor cells.
Electric charge; Phosphatidylcholine; Phosphatidylethanolamine; Phosphatidylinositol; Phosphatidylserine; Renal cancer
A mathematic–physical model of the interaction between cell membrane bilayer leaflets is proposed based on the Casimir effect in dielectrics. This model explains why the layers of a lipid membrane gently slide one past another rather than penetrate each other. The presented model reveals the dependence of variations in the free energy of the system on the membrane thickness. This function is characterized by the two close minima corresponding to the different levels of interdigitation of the lipids from neighbor layers. The energy barrier of the compressing transition between the predicted minima is estimated to be 5.7 kT/lipid, and the return energy is estimated to be 3.1 kT/lipid. The proposed model enables estimation of the value of the membrane elastic thickness modulus of compressibility, which is 1.7 × 109 N/m2, and the value of the interlayer friction coefficient, which is 1.9 × 108 Ns/m3.
Cell membrane; Lipid bilayer; Casimir effect