Transient Increase In Membrane Permeability Research Articles

RNA interference reveals the escape response mechanism of Paramecium to mechanical stimulation.

In Paramecium, a mechanical stimulus applied to the posterior portion of the cell causes a transient increase in membrane permeability to potassium ions, transiently rendering the membrane in a hyperpolarized state. Hyperpolarization causes a transient increase in Cyclic adenosine monophosphate (cAMP) concentration in the cilia, resulting in a transient fast-forward swimming of the cell. Schultz and coworkers (1992) reported that a unique adenylate cyclase (AC)-coupled potassium channel is involved in the reaction underlying this response, which is known as the "escape response." However, the AC responsible for this reaction remains to be identified. Moreover, the molecular linkage between mechanoreception and AC activation has not been elucidated adequately. Currently, we can perform an efficient and simple gene-knockdown technique in Paramecium using RNA interference (RNAi). Paramecium is one of the several model organisms for which whole-genome sequences have been elucidated. The RNAi technique can be applied to whole genome sequences derived from the Paramecium database (ParameciumDB) to investigate the types of proteins that elicit specific biological responses and compare them with those of other model organisms. In this review, we describe the applications of the RNAi technique in elucidating the molecular mechanism underlying the escape response and identifying the AC involved in this reaction. The findings of this study highlight the advantages of the RNAi technique and ParameciumDB.

Exposure to high-frequency electromagnetic field triggers rapid uptake of large nanosphere clusters by pheochromocytoma cells.

BackgroundEffects of man-made electromagnetic fields (EMF) on living organisms potentially include transient and permanent changes in cell behaviour, physiology and morphology. At present, these EMF-induced effects are poorly defined, yet their understanding may provide important insights into consequences of uncontrolled (e.g., environmental) as well as intentional (e.g., therapeutic or diagnostic) exposure of biota to EMFs. In this work, for the first time, we study mechanisms by which a high frequency (18 GHz) EMF radiation affects the physiology of membrane transport in pheochromocytoma PC 12, a convenient model system for neurotoxicological and membrane transport studies.Methods and resultsSuspensions of the PC 12 cells were subjected to three consecutive cycles of 30s EMF treatment with a specific absorption rate (SAR) of 1.17 kW kg−1, with cells cooled between exposures to reduce bulk dielectric heating. The EMF exposure resulted in a transient increase in membrane permeability for 9 min in up to 90 % of the treated cells, as demonstrated by rapid internalisation of silica nanospheres (diameter d ≈ 23.5 nm) and their clusters (d ≈ 63 nm). In contrast, the PC 12 cells that received an equivalent bulk heat treatment behaved similar to the untreated controls, showing lack to minimal nanosphere uptake of approximately 1–2 %. Morphology and growth of the EMF treated cells were not altered, indicating that the PC 12 cells were able to remain viable after the EMF exposure. The metabolic activity of EMF treated PC 12 cells was similar to that of the heat treated and control samples, with no difference in the total protein concentration and lactate dehydrogenase (LDH) release between these groups.ConclusionThese results provide new insights into the mechanisms of EMF-induced biological activity in mammalian cells, suggesting a possible use of EMFs to facilitate efficient transport of biomolecules, dyes and tracers, and genetic material across cell membrane in drug delivery and gene therapy, where permanent permeabilisation or cell death is undesirable.

Asynchronous functional, cellular and transcriptional changes after a bout of eccentric exercise in the rat.

Thirty eccentric contractions (ECs) were imposed upon rat dorsiflexors (n = 46) by activating the peroneal nerve and plantarflexing the foot ~40 deg, corresponding to a sarcomere length change over the range 2.27-2.39 microm for the tibialis anterior and 2.52-2.66 microm for the extensor digitorum longus. Animals were allowed to recover for one of 10 time periods ranging from 0.5 to 240 h, at which time muscle contractile properties, immunohistochemical labelling and gene expression were measured. Peak isometric torque dropped significantly by ~40 % from an initial level of 0.0530 +/- 0.0009 Nm to 0.0298 +/- 0.0008 Nm (P < 0.0001) immediately after EC, and then recovered in a linear fashion to control levels 168 h later. Immunohistochemical labelling of cellular proteins revealed a generally asynchronous sequence of events at the cellular level, with the earliest event measured being loss of immunostaining for the intermediate filament protein, desmin. Soon after the first signs of desmin loss, infiltration of inflammatory cells occurred, followed by a transient increase in membrane permeability, manifested as inclusion of plasma fibronectin. The quantitative polymerase chain reaction (QPCR) was used to measure transcript levels of desmin, vimentin, embryonic myosin heavy chain (MHC), myostatin, myoD and myogenin. Compared to control levels, myostatin transcripts were significantly elevated after only 0.5 h, myogenic regulatory factors significantly elevated after 3 h and desmin transcripts were significantly increased 12 h after EC. None of the measured parameters provide a mechanistic explanation for muscle force loss after EC. Future studies are required to investigate whether there is a causal relationship among desmin loss, increased cellular permeability, upregulation of the myoD and desmin genes, and, ultimately, an increase in the desmin content per sarcomere of the muscle.

Pathological changes of isolated spinal cord axons in response to mechanical stretch

White matter strips extracted from adult guinea-pig spinal cords were maintained in vitro and studied physiologically using a double sucrose gap technique and anatomically using a horseradish peroxidase assay. The amplitude of compound action potentials was monitored continuously before, during, and after elongation. Three types of conduction blocks resulting from stretch injury were identified: an immediate, spontaneously reversible component, which may result from a transient increase in membrane permeability and consequent disturbance of ionic distribution; a second component that was irreversible within 30–60 min of recording, perhaps resulting from profound axolemmal disruption; and a third component, which may be due to perturbation of the myelin sheath, that was reversible with application of 100 μM of the potassium channel blocker, 4-aminopyridine. The intensity of the conduction deficits correlated with the extent of initial stretch over a full range of severity. Stimulus–response data indicate that mechanical damage to axons in stretch was evenly distributed across the caliber spectrum. Morphological examinations revealed that a small portion of axons exhibited membrane damage at 2 min following stretch and appeared to be largely sealed at 30 min after injury. Further, in the entire length of the cord strip subjected to stretch, axons closer to the surface were found to be more likely to suffer membrane damage, which distinguished stretch injury from compression injury. In summary, we have developed an in vitro model of axonal stretch that provides the ability to monitor changes in the properties of central myelinated axons following stretch injury in the absence of pathological variables related to vascular damage. This initial investigation found no evidence of secondary deterioration of axons in the first 30 min after stretch in vitro, although there was evidence of both transient and lasting physiological and anatomical damage to axons and their myelin sheaths.

Retinal spreading depression induced by photoactivation: involvement of free radicals and potassium

The threshold for spreading depression (SD) in chicken retina elicited by rose bengal photoactivation was raised by Trolox (water soluble vitamin E) suggesting the participation of reactive oxygen species in SD. The typical increases in K + concentration associated with SD were preceded by small K + oscillations that were more sensitive to photoactivation than was SD. It is hypothesised that all these phenomena could be accounted for by a free-radical mediated transient increase in membrane permeability and that this may be relevant to ischemic brain damage.

Compression injury of mammalian spinal cord in vitro and the dynamics of action potential conduction failure.

1. White matter strips from the ventral spinal cord of adult guinea pigs were isolated in vitro, and their electrophysiological characteristics and response to controlled focal compression injury were examined. A double sucrose gap technique was used for stimulation and recording at opposite ends of a 12.5 mm-diam central well superfused with oxygenated Krebs solution. 2. The compound action potential recorded with the sucrose gap was similar in form to single fiber potentials recorded with intra-axonal electrodes, including the presence of a prolonged depolarizing afterpotential. 3. Three types of conduction block resulting from compression were identified: an immediate, spontaneously reversible component, which may result from a transient increase in membrane permeability and consequent disturbance of ionic distribution; a second component that was irreversible within 1-2 h of recording, perhaps resulting from complete axolemmal disruption; and a third component, which may have been due to disruption of the myelin sheath, that appeared to be reversible with application of 10-100 microM of the potassium channel blocker 4-aminopyridine. 4. Conduction deficits--decreased amplitude and increased latency of the compound potential--were stable between 5 and 60 min postinjury, and their intensity corellated with the extent of initial compression over a full range of severity. 5. Stimulus-response data indicate that mechanical damage to axons in compression was evenly distributed across the caliber spectrum, suggesting that the susceptibility of large caliber axons seen histopathologically after injury in vivo may be based on delayed, secondary processes. 6. The model provides the ability to monitor changes in the properties of central myelinated axons after compression injury in the absence of pathological variables related to vascular damage. This initial investigation found no evidence of secondary deterioration of axons in the 1st h after injury, although there was evidence of both transient and lasting mechanical damage to axons and their myelin sheaths.

Medical applications and bioeffects of extracorporeal shock waves

Lithotripter shock waves are pressure pulses of microsecond duration with peak pressures of 35–120 MPa followed by a tensile wave. They are an established treatment modality for kidney and gallstone disease. Further applications are pancreatic and salivary stones, as well as delayed fracture healing. The latter are either on their way to become established treatments or are currently under investigation. Shock waves generate tissue damage as a side effect which has been extensively investigated in the kidney, the liver, and the gallbladder. The primary adverse effects are local destruction of blood vessels, bleedings, and formation of blood clots in vessels. Investigations on the mechanism of shock wave action revealed that lithotripters generate cavitation both in vitro and in vivo. An increase in tissue damage at higher pulse administration rates, and also at shock wave application with concomitant gas bubble injection suggested that cavitation is a major mechanism of tissue damage. Disturbances of the heart rhythm and excitation of nerves are further biological effects of shock waves; both are probably also mediated by cavitation. On the cellular level, shock waves induce damage to cell organelles; its extent is related to their energy density. They also cause a transient increase in membrane permeability which does not lead to cell death. Administered either alone or in combination with drugs, shock waves have been shown to delay the growth of small animal tumors and even induce tumor remissions. While the role of cavitation in biological effects is widely accepted, the mechanism of stone fragmentation by shock waves is still controversial. Cavitation is detected around the stone and hyperbaric pressure suppresses fragmentation; yet major cracks are formed early before cavitation bubble collapse is observed. The latter has been regarded as evidence for a direct shock wave effect.

Transient increase in membrane permeability of L1210 cells upon exposure to lithotripter shock waves in vitro.

Transient increase in membrane permeability of L1210 cells upon exposure to lithotripter shock waves in vitro.

Complement induces a transient increase in membrane permeability in unlysed erythrocytes.

The effects of low concentrations of human serum on antibody-sensitized sheep erythrocytes (EA) were studied. We report that exposure to low concentrations of serum induced a large but transient increase in the membrane permeability of those EA that do not lyse. This change in the permeability of the erythrocyte membrane resulted in net uptake of Na+ and decrease in cell K+, without affecting the total internal cation content. Although exposure to serum also allowed for net uptake of larger molecules like L-glucose, it did not lead to cell swelling. Experiments with sera genetically deficient in one of the terminal complement components showed that C8, but not C9, was required to produce the observed change in membrane permeability. Therefore, we propose that the C5b-8 complex can mediate the transient increase in permeability observed in unlysed erythrocytes during complement activation by whole serum.

Cytoplasmic proteins, association with phospholipid vesicles and its dependency on cholesterol.

Since the plasticity of the plasma membrane is correlated with changes in its dynamic behavior and biological functions, the content of cholesterol in this membrane is thought to have an important role in the regulation of cell metabolism. The research reported here was done to clarify the role of cholesterol in the interaction of the innersurface of plasma membrane with cytoplasmic proteins in relation to the regulatory mechanism of signal transduction. Many cytoplasmic proteins of EATC or rat liver cells were found to associate with DPPC liposomes and they induced a transient increase in membrane permeability at the phase transition temperature of the liposomal lipid. The association and the permeability increase were inhibited by the introduction of cholesterol into DPPC liposomes, and the sensitivity of individual proteins to the action of cholesterol differed. F-actin, but not G-actin, associated selectively with the liposomes. Also, the main endothermic peak of the unilamellar DPPC liposomes was shifted from 37 degrees C to 43 degrees C by this protein association, evidence of transformation from a unito multilamellar structure. The introduction of cholesterol into the liposomal membrane caused a reduction in the energy content of the phase transition and in the inhibition of protein-membrane interaction. We concluded that the cholesterol in the plasma membrane contributes to the regulation of cell surface signal transduction.

Formyl peptide stimulation of superoxide anion release from lung macrophages: sodium and potassium involvement.

We examined the role of the monovalent cations Na+ and K+ in the events encompassing the release of O-2 by alveolar macrophages after stimulation with formyl methionyl phenylalanine (FMP). This was accomplished by determining the effect of changing the extracellular [Na+] and/or [K+] on FMP-stimulated O-2 production; and measuring 22Na+, 42K+ and 86Rb+ influx and efflux and intracellular [K+] for control and FMP-stimulated alveolar macrophages. Stimulated O-2 production was relatively insensitive to changes in extracellular K+ or Na+ concentrations until the [Na+] was decreased below 35 mM. At 4 mM [Na+], the rate of O-2 production remained at 75% of the maximal rate observed at physiological concentrations of [Na+]. Both influx and efflux of 22Na+ were stimulated above control rates by FMP. The increased rates of fluxes lasted for a few minutes suggesting a transient increase in membrane permeability to Na+. Ouabain partially inhibited 22Na+ efflux but had no effect on O-2 release. The influx of 86Rb+ and 42K+ was not altered by the addition of FMP but was virtually abolished in the presence of 10 microM ouabain or 1 mM quinine. In the presence of extracellular calcium, FMP-stimulated a prolonged (greater than 20 minutes) increase in 86Rb+ or 42K+ efflux which was inhibitable by 1 mM quinine. In the absence of extracellular calcium, FMP stimulation of K+ efflux was greatly diminished and was not affected by quinine, although quinine still inhibited O-2 production under these conditions. It was also observed that there was a loss of intracellular K+ when cells were stimulated by FMP in the presence of Ca+2, but not in the absence of Ca+2. Taken together, these results suggest a minimal direct role, if any, for K+ in the events that lead to FMP-stimulated O-2 release by alveolar macrophages.

Stimulation of 45CA efflux from rat lacrimal gland slices by carbachol and epinephrine

The effects of carbachol (10 −5M) and epinephrine (10 −5M) on efflux of 45Ca from rat exorbital lacrimal gland slices were examined. Both carbachol and epinephrine stimulated a transient release of 45Ca from the tissue. The quantity of Ca released was estimated to be of the order of 0.5 μmol/g. Release of 45Ca by one agonist prevented subsequent release of 45Ca by a different agonist. These data support the hypothesis put forth previously that in the lacrimal gland muscarinic or α-adrenergic receptor activation causes a transient increase in membrane permeability to K by triggering the release of a sizable intracellular pool of Ca common to both receptors.

Ionic mechanisms controlling behavioral responses of paramecium to mechanical stimulation.

A mechanical stimulus applied to the anterior part of Paramecium causes a transient increase in membrane permeability to calcium. This permits a calcium current to flow into the cell, causing the membrane potential to approach the equilibrium level for calcium. The transient depolarization which results elicits a reversal in the direction of ciliary beat. When the organisms are free-swimming this is seen as the reversed locomotion of Jennings' "avoiding reaction." In contrast, a mechanical stimulus applied to the posterior part results in increased permeability to potassium ions, and hence an outward potassium current. The hyperpolarization which results causes an increase in the frequency of ciliary beat in the normal direction. In free-swimming specimens this is seen as an increase in the velocity of forward locomotion.