Proteolytic cleavages in alpha 1-antitrypsin and microheterogeneity

Antitrypsin was resolved into two pools by ion-exchange chromatography. Pool 2 contained three anodal isoinhibitors and an N-terminal sequence identical with the one found by others. Pool 1 contained, in addition to the anodal ones two cathodal isoinhibitors as well. The sequencing data of Pool 1 indicate that the cathodal proteins are formed from the anodals by a cleavage of the Gly5-Asp6 bond in the molecule.     

The purification and properties of human alpha1-antitrypsin (alpha1-antiprotease), variant Z.

After three stages of preliminary purification, variant Z was chromatographed on a DEAE-cellulose column. Upon elution with a linearly increasing concentration of NaCl, variant Z was recovered in two separate peaks, the first of which contained 81% and the second 19% of the total. The preparation corresponding to the first peak was homogeneous by various criteria. The trypsin and chymotrypsin inhibiting capacities and the specific antigenic activity of the preparation were nearly the same as those of an authentic sample of variant M. Variant Z contained 8 or 9 more gycine residues than variant M, but no appreciable difference was found between their carbohydrate contents. By analytical isoelectrofocusing the isoinhibitors of purified variant Z overlapped with those in the plasma of the donor and were cathodal to, but partially overlapped with purified variant M. After desialysation, the overlap between the different variants became complete, but variant Z contained a larger proportion of cathodal and smaller proportion of anodal components than variant M. Both variants formed five distinct isoinhibitor-protease complexes after incubation with trypsin and chymotrypsin and the corresponding complexes in the different variants completely coincided.     

Modification of the isoinhibitors of human serum alpha 1-proteinase inhibitor (alpha 1-antitrypsin) by pancreatic proteases

Due to the action of a serum protease, the two most cathodal isoinhibitors of the alpha 1-proteinase inhibitor (alpha 1-PI) are cleaved at the Gly5-Asp6 bond and lack two negative charges. In spite of this, these can bind trypsin and chymotrypsin, showing that the N-terminal pentapeptide is not indispensable for inhibition function. Pancreatic proteases also cleave a bond near the N-terminus in alpha 1-PI, resulting in a loss of two negative charges and a corresponding cathodal shift in the electrofocusing behavior of the isoinhibitors. Trypsin cleaves isoinhibitors near the N-terminus at a large inhibitor excess and unless an additional cleavage takes place, at least two of the new isoinhibitors remain active. An additional cleavage(s), most likely at a distance of 30-40 residues from the C-terminus results in a corresponding decrease of the molecular mass and a loss of inhibition function. Although the C-terminal cleavage peptide does separate from the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it remains associated with it under conditions of polyacrylamide gel isoelectric focusing. Chymotrypsin also cleaved alpha 1-PI near the N-terminus but this could be observed only at protease excess and the modified isoinhibitors did not form complexes with chymotrypsin. The molecular polymorphism of alpha 1-PI is partly explained by the absence of the N-terminal pentapeptide from some of the isoinhibitors.     

Unusual sialilation of three different rare genetic variants of serum DBP: Gc 1A17, Gc 1A16, and Gc 1A11

Summary The proteins of three anodal Gc1 variants, Gc 1A16, 1A11, and 1A17, are characterized by the most acidic isoelectric points observed so far among the different Gc mutants. Stepwise removal of N-acetylneuraminic acid (NANA) by treatment with neuraminidase was performed to estimate the degree of sialilation of these Gc variants. The results indicate that both proteins, the anodal and the cathodal component of these Gc 1 mutants, carry sialic acid residues. This observation is remarkable in so far as usually only the anodal component of the Gc 1 protein contains NANA and only a single residue. From the experiments carried out it can be deduced that Gc 1A16 has two NANA residues in the anodal and one NANA residue in the cathodal component. Gc 1A16 was found in four members of three generations in a Danish family; the variant segregated as a Mendelian trait. More difficult to interprete are the results obtained with the variants Gc 1A11 and Gc 1A17. Gc 1A11 probably has three NANA residues in the anodal and two NANA residues in the cathodal component. Gc 1A11 has been observed in two mother-child pairs and is presumably also a simple genetic trait. Gc 1A17 has also several NANA residues in both Gc proteins; it is suggested that the anodal component has either three or four NANA residues and the cathodal component either two or three NANA residues. Family information on this variant is not yet available.     

Taste transduction mechanism: similar effects of various modifications of gustatory receptors on neural responses to chemical and electrical stimulation in the frog

Responses in the frog glossopharyngeal nerve induced by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCl2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCl at equal current intensities, as chemical stimulation with MgCl2 produced much larger responses than stimulation with NaCl at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-1-naphthalenesulfonate, NiCl2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCl2 resulted in a large response, whereas application of 1 M CaCl2 to the tongue adapted to 50 mM CaCl2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCl2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdCl2 and verapamil) to the perfusing solution of the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCl) and to anodal current with 10 mM NaCl. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.     

The role of passive calcium influx through the cell membrane in galvanotaxis

Passive calcium influx is one of the theories to explain the cathodal galvanotaxis of cells that utilize the electric field to guide their motion. When exposed to an electric field, the intracellular fluid becomes polarized, leading to positive charge accumulation on the cathodal side and negative charge accumulation on the anodal side. The negative charge on the anodal side attracts extracellular calcium ions, increasing the anodal calcium concentration, which is supposed to decrease the mobile properties of this side. Unfortunately, this model does not capture the Ca²⁺ dynamics after its presentation to the intracellular fluid. The ions cannot permanently accumulate on the anodal side because that would build a potential drop across the cytoplasm leading to an ionic current, which would carry positive ions (not only Ca²⁺) from the anodal to the cathodal part through the cytoplasm. If the cytoplasmic conductance for Ca²⁺ is low enough compared to the membrane conductance, the theory could correctly predict the actual behavior. If the ions move through the cytoplasm at a faster rate, compensating for the passive influx, this theory may fail. This paper contains a discussion of the regimes of validity for this theory.     

Improving Cycling Performance: Transcranial Direct Current Stimulation Increases Time to Exhaustion in Cycling

The central nervous system seems to have an important role in fatigue and exercise tolerance. Novel noninvasive techniques of neuromodulation can provide insights on the relationship between brain function and exercise performance. The purpose of this study was to determine the effects of transcranial direct current stimulation (tDCS) on physical performance and physiological and perceptual variables with regard to fatigue and exercise tolerance. Eleven physically active subjects participated in an incremental test on a cycle simulator to define peak power output. During 3 visits, the subjects experienced 3 stimulation conditions (anodal, cathodal, or sham tDCS—with an interval of at least 48 h between conditions) in a randomized, counterbalanced order to measure the effects of tDCS on time to exhaustion at 80% of peak power. Stimulation was administered before each test over 13 min at a current intensity of 2.0 mA. In each session, the Brunel Mood State questionnaire was given twice: after stimulation and after the time-to-exhaustion test. Further, during the tests, the electromyographic activity of the vastus lateralis and rectus femoris muscles, perceived exertion, and heart rate were recorded. RM-ANOVA showed that the subjects performed better during anodal primary motor cortex stimulation (491 ± 100 s) compared with cathodal stimulation (443 ± 11 s) and sham (407 ± 69 s). No significant difference was observed between the cathodal and sham conditions. The effect sizes confirmed the greater effect of anodal M1 tDCS (anodal x cathodal = 0.47; anodal x sham = 0.77; and cathodal x sham = 0.29). Magnitude-based inference suggested the anodal condition to be positive versus the cathodal and sham conditions. There were no differences among the three stimulation conditions in RPE (p = 0.07) or heart rate (p = 0.73). However, as hypothesized, RM- ANOVA revealed a main effect of time for the two variables (RPE and HR: p < 0.001). EMG activity also did not differ during the test accross the different conditions. We conclude that anodal tDCS increases exercise tolerance in a cycling-based, constant-load exercise test, performed at 80% of peak power. Performance was enhanced in the absence of changes in physiological and perceptual variables.     

Paced monophasic and biphasic waveforms alter transmembrane potentials and metabolism of human fibroblasts

Resting transmembrane potential (TMP) of primary human fibroblast cells was altered in predictable directions by subjecting cell cultures to specific monophasic and biphasic waveforms. Cells electrically stimulated with an anodal pulse resulted in hyperpolarization while a cathodal waveform depolarized the TMP to below that of non-paced control cells. The biphasic waveform, consisting of an anodal pulse followed immediately by an inverse symmetric cathodal pulse, also lessened the TMP similar to that of the cathodal pulse. The effect of short-term pacing on the TMP can last up to 4 h before the potentials equilibrate back to baseline. While subjecting the cells to this electrical field stimulation did not appear to damage the integrity of the cells, the three paced electrical stimulation waves inhibited expansion of the cultures when compared to non-paced control cells. With longer pacing treatments, elongation of the cells and electrotaxis towards the anodal polarity were observed. Pacing the fibroblasts also resulted in modest, yet very statistically significant (and likely underestimated) changes to cellular adenosine-5'-triphosphate (ATP) levels, and cells undergoing anodal and biphasic (anodal/cathodal) stimulation also exhibited altered mitochondrial morphology. These observations indicate an active role of electrical currents, especially with anodal content, in affecting cellular metabolism and function, and help explain accumulating evidence of cellular alterations and clinical outcomes in pacing of the heart and other tissues in general.     

Fish species richness and incidence patterns in isolated and connected stream pools: effects of pool volume and spatial position

I tested the effects of pool size and spatial position (upstream or downstream) on fish assemblage attributes in isolated and connected pools in an upland Oklahoma stream, United States. I hypothesized that there would be fundamental differences between assemblages in these two pool types due to the presence or absence of colonization opportunities. Analyses were carried out at three ecological scales: (1) the species richness of pool assemblages, (2) the species composition of pool assemblages, and (3) the responses of individual species. There were significant species-volume relationships for isolated and connected pools. However, the relationship was weaker and there were fewer species, on average, in isolated pools. For both pool types, species incidences were significantly nested such that species-poor pools tended to be subsets of species-rich pools, a common pattern that ultimately results from species-specific differences in colonization ability and/or extinction susceptibility. To examine the potential importance of these two processes in nestedness patterns in both pool types, I made the following two assumptions: (1) probability of extinction should decline with increasing pool size, and (2) probability of immigration should decline in an upstream direction (increasing isolation). When ordered by pool volume, only isolated pools were significantly nested suggesting that these assemblages were extinction-driven. When ordered by spatial position, only connected pools were significantly nested (more species downstream) suggesting that differences in species-specific dispersal abilities were important in structuring these assemblages. At the individual-species level, volume was a significant predictor of occurrence for three species in isolated pools. In connected pools, two species showed significant position effects, one species showed a pool volume effect, and one species showed pool volume and position effects. These results demonstrate that pool size and position within a watershed are important determinants of fish species assemblage structure, but their importance varies with the colonization potential of the pools. Isolated pool assemblages are similar to the presumed relaxed faunas of montane forest fragments and land bridge islands, but at much smaller space and time scales. Received: 6 December 1996 / Accepted: 10 December 1996     

Changes in annexin (lipocortin) content in human amnion and chorion at parturition.

Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.     

Structural and functional studies of the king salmon, Oncorhynchus tshawytscha, hemoglobins

Vertical starch-gel electrophoresis at pH 8.6 revealed extensive hemoglobin multiplicity with several distinct cathodal and anodal hemoglobin components. Anodal hemoglobin components are present throughout the life cycle of the king salmon. Additional cathodal components are found in the adult fish. Cathodal hemoglobin components exhibited a higher oxygen affinity (P50 = 10.2 mm at 13 degrees C, pH 7.3) than the anodal hemoglobin components (P50 = 21.8 mmHg at 13 degrees C). Oxygen binding of the anodal hemoglobins are sensitive to pH, temperature, organic phosphates (ATP and GTP), as well as, ionic strength; binding of oxygen to the cathodal hemoglobins is independent of pH and not affected by organic phosphates. Anodal hemoglobin components are less resistant to thermal denaturation over the pH 6.0 to 8.0 range. Isothermal urea denaturation of separated anodal and cathodal hemoglobin fractions of the king salmon indicate inherent differences in the stabilization energies of these hemoglobins. Autoxidation of these hemoglobins occurs around pH 7.0 and below, as well as, in the presence of increasing Cl- concentrations.     

Analysis of nerve conduction block induced by direct current

The mechanisms of nerve conduction block induced by direct current (DC) were investigated using a lumped circuit model of the myelinated axon based on Frankenhaeuser–Huxley (FH) model. Four types of nerve conduction block were observed including anodal DC block, cathodal DC block, virtual anodal DC block, and virtual cathodal DC block. The concept of activating function was used to explain the blocking locations and relation between these different types of nerve block. Anodal/cathodal DC blocks occurred at the axonal nodes under the block electrode, while virtual anodal/cathodal DC blocks occurred at the nodes several millimeters away from the block electrode. Anodal or virtual anodal DC block was caused by hyperpolarization of the axon membrane resulting in the failure of activating sodium channels by the arriving action potential. Cathodal or virtual cathodal DC block was caused by depolarization of the axon membrane resulting in inactivation of the sodium channel. The threshold of cathodal DC block was lower than anodal DC block in most conditions. The threshold of virtual anodal/cathodal blocks was about three to five times higher than the threshold of anodal/cathodal blocks. The blocking threshold was decreased with an increase of axonal diameter, a decrease of electrode distance to axon, or an increase of temperature. This simulation study, which revealed four possible mechanisms of nerve conduction block in myelinated axons induced by DC current, can guide future animal experiments as well as optimize the design of electrodes to block nerve conduction in neuroprosthetic applications.     

Task-specific effects of tDCS-induced cortical excitability changes on cognitive and motor sequence set shifting performance

In this study, we tested the effects of transcranial Direct Current Stimulation (tDCS) on two set shifting tasks. Set shifting ability is defined as the capacity to switch between mental sets or actions and requires the activation of a distributed neural network. Thirty healthy subjects (fifteen per site) received anodal, cathodal and sham stimulation of the dorsolateral prefrontal cortex (DLPFC) or the primary motor cortex (M1). We measured set shifting in both cognitive and motor tasks. The results show that both anodal and cathodal single session tDCS can modulate cognitive and motor tasks. However, an interaction was found between task and type of stimulation as anodal tDCS of DLPFC and M1 was found to increase performance in the cognitive task, while cathodal tDCS of DLPFC and M1 had the opposite effect on the motor task. Additionally, tDCS effects seem to be most evident on the speed of changing sets, rather than on reducing the number of errors or increasing the efficacy of irrelevant set filtering.     

Retinol-binding protein from human urine and its interaction with retinol and prealbumin

In freshly collected urine from a patient with glomerulotubular proteinuria there were two bands which contained retinol-binding proteins. The cathodal band showed fluorescence in the ultraviolet. After extraction with organic solvents only the anodal non-fluorescent band remained. After addition of an excess retinol only one band remained which by mobility corresponded to the cathodal band.The anodal of the two bands was therefore probably the apo form and the cathodal the holo form of the same retinol-binding protein. Their proportions, determined by densitometric scanning were approximately 4/1 (anodal/cathodal band). More than 85% of the retinol-binding protein in the urine bound to prealbumin-Sephrose. The apo retinol-binding protein from urine had the same electrophoretic mobility on agarose gel el-ctrophoresis and the same pattern on isoelectric focusing as an retinol-binding protein prepared from serum. The carboxy-terminal amino acid sequence of the retinol-binding protein from freshly collected urine that bound to prealbumin-Sepharose, was -Arg-Leu. The amino-terminal sequence was Glu-Arg-Asp-Cys-Arg-Val-Ser-X-Phe-Arg-Val-Lys-Glu-Asn-Phe-Asp-Lys-Ala-Arg-Phe-X-Gly-Thr-Trp-Tyr-. This sequence and the amino acid composition are compatible with the view that the retinol-binding protein in urine is the same as in plasma.     

Multi-Session Transcranial Direct Current Stimulation (tDCS) Elicits Inflammatory and Regenerative Processes in the Rat Brain

Transcranial direct current stimulation (tDCS) is increasingly being used in human studies as an adjuvant tool to promote recovery of function after stroke. However, its neurobiological effects are still largely unknown. Electric fields are known to influence the migration of various cell types in vitro, but effects in vivo remain to be shown. Hypothesizing that tDCS might elicit the recruitment of cells to the cortex, we here studied the effects of tDCS in the rat brain in vivo. Adult Wistar rats (n = 16) were randomized to either anodal or cathodal stimulation for either 5 or 10 consecutive days (500 µA, 15 min). Bromodeoxyuridine (BrdU) was given systemically to label dividing cells throughout the experiment. Immunohistochemical analyses ex vivo included stainings for activated microglia and endogenous neural stem cells (NSC). Multi-session tDCS with the chosen parameters did not cause a cortical lesion. An innate immune response with early upregulation of Iba1-positive activated microglia occurred after both cathodal and anodal tDCS. The involvement of adaptive immunity as assessed by ICAM1-immunoreactivity was less pronounced. Most interestingly, only cathodal tDCS increased the number of endogenous NSC in the stimulated cortex. After 10 days of cathodal stimulation, proliferating NSC increased by ∼60%, with a significant effect of both polarity and number of tDCS sessions on the recruitment of NSC. We demonstrate a pro-inflammatory effect of both cathodal and anodal tDCS, and a polarity-specific migratory effect on endogenous NSC in vivo. Our data suggest that tDCS in human stroke patients might also elicit NSC activation and modulate neuroinflammation.     

Human alpha 1-antitrypsin genetic polymorphism: PI N subtypes

Three new genetic variants (PI types) of alpha 1-antitrypsin are described. They have been compared to previously described phenotypes by several techniques including narrow pH range isoelectric focusing in ultrathin polyacrylamide gels. In this system, the relevant alpha 1-antitrypsin gel bands, identified by crossed immunoelectrophoresis, focused between PI M2, the most cathodal PI M subtype, and PI P BUD, the most anodal PI P subtype. They were therefore considered to be PI N subtypes. Two of them, PI N GRO and PI N YER, could not be separated by isoelectric focusing, but gave a different pattern in agarose gel electrophoresis. None of the new alleles seemed to be associated with disease. The high resolving power of isoelectric focusing is emphasized with respect to the information it may provide concerning amino acid substitutions, while the use of other techniques proved to be of utmost importance in the differentiation of other variants showing similar isoelectric points.     

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat-1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen--Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat-1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed beta 1-integrin-containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcranial Electrical Stimulation over Dorsolateral Prefrontal Cortex Modulates Processing of Social Cognitive and Affective Information

Recent neurofunctional studies suggested that lateral prefrontal cortex is a domain-general cognitive control area modulating computation of social information. Neuropsychological evidence reported dissociations between cognitive and affective components of social cognition. Here, we tested whether performance on social cognitive and affective tasks can be modulated by transcranial direct current stimulation (tDCS) over dorsolateral prefrontal cortex (DLPFC). To this aim, we compared the effects of tDCS on explicit recognition of emotional facial expressions (affective task), and on one cognitive task assessing the ability to adopt another person’s visual perspective. In a randomized, cross-over design, male and female healthy participants performed the two experimental tasks after bi-hemispheric tDCS (sham, left anodal/right cathodal, and right anodal/left cathodal) applied over DLPFC. Results showed that only in male participants explicit recognition of fearful facial expressions was significantly faster after anodal right/cathodal left stimulation with respect to anodal left/cathodal right and sham stimulations. In the visual perspective taking task, instead, anodal right/cathodal left stimulation negatively affected both male and female participants’ tendency to adopt another’s point of view. These findings demonstrated that concurrent facilitation of right and inhibition of left lateral prefrontal cortex can speed-up males’ responses to threatening faces whereas it interferes with the ability to adopt another’s viewpoint independently from gender. Thus, stimulation of cognitive control areas can lead to different effects on social cognitive skills depending on the affective vs. cognitive nature of the task, and on the gender-related differences in neural organization of emotion processing.     

Effect of sodium,potassium, and calcium ions on electroreceptor function in catfish

Impulses from single electroreceptors (small pit organs) of catfish (Ictalurus nebulosus) were recorded during stimulation by square pulses. Solutions with different concentrations of potassium, sodium, and calcium ions were applied to the pore of the receptor. Solutions with a low CaCl₂ concentration did not alter the responses of the receptor. Calcium ions in concentrations of over 5 mM increased the threshold of the response to electrical stimulation. The threshold to anodal stimulation was increased in solutions of 2 mM sodium and potassium and no response was given to a cathodal stimulus. The effect of 2 mM solutions of NaCl and KCl was abolished by the addition of 0.4 mM CaCl₂ or by application of a long anodal stimulus of high intensity (10⁻⁸∓10⁻⁷ A/mm²). Increasing the potassium ion concentration to 10–20 mM restored normal receptor function but a further increase led to elevation of the threshold. The action of an electric current is compared with the action of the ions.     

Effects of biphasic and monophasic electrical stimulation on mitochondrial dynamics,cell apoptosis,and cell proliferation

Currently, electrical stimulation (ES) is used to induce changes in various tissues and cellular processes, but its effects on mitochondrial dynamics and mechanisms are unknown. The aim of this study was to compare the effects of monophasic and biphasic, anodal, and cathodal ES on apoptosis, proliferation, and mitochondrial dynamics in neuroblastoma SH-SY5Y cells. Cells were cultured and treated with ES. Alamar blue assay was performed to measure cell proliferation. The proteins expression of apoptotic-related proteins Bcl-2 associated X (Bax), B cell lymphoma 2 (Bcl-2), optic-atrophy-1 (OPA1), mitofusin2 (Mfn2), phosphorylated dynamin-related protein 1 at serine 616 (p-DRP1), and total dynamin-related protein 1 (Total-DRP1) were also determined. The results showed that monophasic anodal and biphasic anodal/cathodal (Bi Anod) ES for 1 hr at 125 pulses per minute (2.0 Hz) produced the most significant increase in cell proliferation. In addition, monophasic anodal and Bi Anod ES treated cells displayed a significant increase in the levels of anti-apoptotic protein Bcl-2, whereas the Bax levels were not changed. Moreover, the levels of Mfn2 were increased in the cells treated by Bi Anod, and OPA1 was increased by monophasic anodal and Bi Anod ES, indicating increased mitochondrial fusion in these ES-treated cells. However, the levels of mitochondrial fission indicated by DRP1 remained unchanged compared with non-stimulated cells. These findings were confirmed through visualization of mitochondria using Mitotracker Deep Red, demonstrating that monophasic anodal and Bi Anod ES could induce pro-survival effects in SH-SY5Y cells through increasing cell proliferation and mitochondrial fusion. Future research is needed to validate these findings for the clinical application of monophasic anodal and Bi Anod ES.