Extracellular Solution Research Articles

Green Synthesis of Gold Nanoparticles: A Novel, Environment-Friendly, Economic, Safe Approach

Nanoparticles can be synthesised using a variety of methods. These approaches, on the other hand, are connected with the development of undesired byproducts that are both harmful and expensive. As a result, several attempts are being undertaken to develop unique, cost-effective, safe, and dependable "green" techniques for producing desirable nanoparticles. To develop a novel, environment-friendly, economic, safe approach to the synthesis of gold nanoparticles via the biological entity. Addition of aqueous gold chloride solution to the microwave-exposed aqueous extracellular Cassia tora leaf extract yielded poly shaped gold nanoparticles. The UV-vis. spectroscopic investigations are led to notice and affirm the formation of nanoparticles. FTIR studies are performed to affirm the role of a biomolecule in stabilizing the nanoparticles. X-beam diffraction study is utilized to affirm the crystalline nature of nanoparticles. The elemental characterization of the samples is regulated by EDX studies. The size and morphology of the synthesized nanoparticles are explored using HR-TEM analysis and FESEM. It is seen that the flavonoids which are separated during microwave warming of extracellular solution of the cassia tora leaves are liable for the biosynthesis of gold nanoparticles. The nanoparticle was noted to be well dispersed and polyshaped with a 20-60 nm range. The leaf extract based preparation of AuNP is more gainful since leaf is used instead of microorganism as many of the issues like pathogenicity, procedural maintenance of hygiene of cell culture and labor efforts can be overcome. The presence of flavonoids in the leaf was discovered by the examination of produced nanoparticles, suggesting that they may have fulfilled both reduction and stabilisation activities. The presented approach can be inferred to be cost-effective, environmentally friendly, and capable of manufacturing nanoparticles with desired physical and pharmacological properties.

Theoretical approaches to determination of optimal cryopreservation regimens for cell spheroids of different cultivation terms

Background: Three-dimensional culture systems are unique platforms for studying complex biological processes in vitro. Cell-cell and cell-extracellular matrix interactions form a communication network of biochemical and mechanical signals, bring spheroids (SP) closer to native tissues and significantly distinguish them from monolayer cultures. It is important for cell technologies to develop methods for cryopreservation of 3D cultures, that allows creating the stocks of valuable cell samples, save time and materials, and prevent the loss of cultures due to technical failures, contamination, phenotype drift and aging. Objectives: Development of approaches to cellular spheroids cryopreservation. Determination of the permeability parameters of L929 cells spheroids at different cultivation periods for the theoretical assessment of optimal freezing regimens. Materials and methods: We have used L929 cells, which form SPs of different diameters and can be maintained for a long time in 3D conditions. To determine the integral filtration Lp and permeability for DMSO kp coefficients for SP at different periods of cultivation, the volumetric method was used. The study of the changes in the spheroids volume in time was carried out with a confocal microscope LSM 510 META. The numerical values of the integral SF permeability coefficients were determined by approximating the experimental data on the change in the relative volume of the SP versus the exposure time in the test solution with theoretical curves calculated on the basis of a physical and mathematical model for passive mass transfer between the spheroid and the environment, provided that they coincide as much as possible. Prediction of the osmotic behavior of spheroids under cooling conditions was carried out based on the differential equation describing the kinetics of changes in the relative cell volume during extracellular crystallization of a cryoprotective solution, substituting determined values of integral permeability coefficients Lp and kp and activation energies EAL and EAk into the model equations. The kinetics of changes in the extracellular solution concentration during freezing was set analytically by approximating the phase melting diagram of the DMSO solution. Results: The filtration and permeability for DMSO molecules coefficients in SP were determined and their significant decrease with a cultivation duration was shown. The activation energy values for the penetration of water and DMSO molecules into the SP were calculated and their dependence on the cultivation time was determined. Proceeding from the determined parameters of permeability, the dynamic of changes in the volume of SPs for different periods of cultivation at different rates of cooling was calculated. Conclusions: The optimal cooling modes of SP from L929 cells were in theory determined: for 7 days of cultivation — 1,5-2 °C/min with cooling to -80°C and subsequent immersion in nitrogen; for 14 and 21 days of cultivation — 0.5 °C/min to -40°C and subsequent immersion in nitrogen.

Prefrontal cortex pyramidal neurons express functional Nav1.8 tetrodotoxin-resistant sodium currents.

It has been repeatedly proved that Nav1.8tetrodotoxin (TTX)-resistant sodium currents are expressed in peripheral sensory neurons where they play important role in nociception. There are very few publications that show the presence of TTX-resistant sodium currents in central neurons. The aim of this study was to assess if functional Nav1.8TTX-resistant sodium currents are expressed in prefrontal cortex pyramidal neurons. All recordings were performed in the presence of TTX in the extracellular solution to block TTX-sensitive sodium currents. The TTX-resistant sodium current recorded in this study was mainly carried by the Nav1.8sodium channel isoform because the Nav1.9 current was inhibited by the -65mV holding potential that we used throughout the study. Moreover, the sodium current that we recorded was inhibited by treatment with the selective Nav1.8 inhibitor A-803467. Confocal microscopy experiments confirmed the presence of the Nav1.8 α subunit in prefrontal cortex pyramidal neurons. Activation and steady state inactivation properties of TTX-resistant sodium currents were also assessed in this study and they were similar to activation and inactivation properties of TTX-resistant sodium currents expressed in dorsal root ganglia (DRG) neurons. Moreover, this study showed that carbamazepine (60µM) inhibited the maximal amplitude of the TTX-resistant sodium current. Furthermore, we found that carbamazepine shifts steady state inactivation curve of TTX-resistant sodium currents toward hyperpolarization. This study suggests that the Nav1.8TTX-resistant sodium channel is expressed not only in DRG neurons, but also in cortical neurons and may be molecular target for antiepileptic drugs such as carbamazepine.

The Mitochondrial Ca2+ import complex is altered in ADPKD

Mutations in either of the polycystic kidney disease genes, PKD1 or PKD2, engender the growth of cysts, altering renal function. Cystic growth is supported by major changes in cellular metabolism, some of which involve the mitochondrion, a major storage site for Ca2+ and a key organelle in cellular Ca2+ signaling. The goal here was to understand the role of components of the mitochondrial Ca2+ uptake complex in PC1-mutant cells in autosomal dominant polycystic kidney disease (ADPKD). We found that the mitochondrial Ca2+ uniporter (MCU) and voltage-dependent anion channels 1& 3 (VDAC) were down-regulated in different mouse and cell models of ADPKD along with the Ca2+-dependent enzyme, pyruvate dehydrogenase phosphatase (PDHX). The release of Ca2+ from the endoplasmic reticulum, and Ca2+ uptake by the mitochondria were upregulated in PC1(polycystin)-null cells. We also observed an enhanced staining with MitoTracker Red CMXRos in PC1-null cultured cells than in PC1-containing cells and a substantially higher increase in response to ER Ca2+ release. Increased colocalization of the Ca2+ sensitive dye, rhodamine2, with MitoTracker Green suggested an increase Ca2+ entry into the mitochondria in PC1 null cells subsequent to Ca2+ release from the ER or from Ca2+ entry from the extracellular solution. These data clearly demonstrate abnormal release of Ca2+ by the ER and corresponding alterations in Ca2+ uptake by the mitochondria in PC1−null cells. Importantly, inhibiting mitochondrial Ca2+ uptake with the specific inhibitor Ru360 inhibited cyst growth and altered both apoptosis and cell proliferation. We further show that the decrease in mitochondrial proteins and abnormally high Ca2+ signaling can be reversed by application of the cystic fibrosis (CFTR) corrector, VX-809. We conclude that enhanced Ca2+ signaling and alterations in proteins association with the mitochondrial Ca2+ uptake complex are associated with malfunction of PC1. Finally, our results identify novel therapeutic targets for treating ADPKD.

Abstract 10782: Optimization of Preservation Solution and Temperature of Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Patch for Regenerative Therapy

Background: Regenerative therapy for heart failure is being practical as a new treatment. Since cell products contain living cells, it is important to apply the storage method according to the characteristics of the product in order to maintain its structure and function. A lot of studies have been done on suitable storage solutions for organs, and organ storage solutions may also be effective for cell product preservation, especially tissues with three-dimensional structures. In this study, we investigated the optimal storage conditions for human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) patches. Methods and Results: We evaluated the properties of iPSC-CM patches after storage in cell culture buffer solution (BS), intracellular fluid type organ preservation solution A (PSA), and extracellular fluid type organ preservation solution B (PSB) and under room temperature or 4°C. Although the patches maintained their sheet-like morphology up to 120hrs , the sarcomere structure was disrupted, and the viability decreased over time after 48hrs of storage in BS at room temperature. Myocardial contraction/relaxation velocity and cytokine production after reculture of the stored patches showed a significant decrease after 96hrs of storage in BS at room temperature. We tested whether PSA and PSB improve the preservation of the patches compared to the BS in 48hrs storage. As a result, contraction velocity after reculture was comparable for PSA (17.1±4.4 μm/sec), PSB (10.0±0.9 μm/sec) and BS (12.9±1.4μm/sec). However, cell viability after storage was significantly decreased in PSA (13.9±19.9%) and PSB (68.4±26.1%) compared to BS (90.6±4.1%). Although sarcomere structure was maintained in BS, it was disrupted in PSA and PSB. No improvement was observed in contraction velocity, cell viability and sarcomere structure in PSA at 4°C compared to BS at room temperature. Conclusions: The iPSC-CM patches maintained their structure and function for up to 48 hrs under storage conditions in BS at room temperature. The buffer solution, intracellular or extracellular fluid type organ preservation solution have different ionic balances, suggesting that the optimal storage conditions differ according to tissue structure and cell type.

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer.

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F - containing internal solution together with a Ca 2+ containing external solution - so that a CaF 2 crystal forms when the two solutions meet which 'plugs the holes' to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F - ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF 2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

Physicochemical properties of brand and generic infusion fluid preparations (Part 3): Investigation of type 1 hypotonic infusion fluids.

In Japan, the increasing use of generic drugs has led to a reduction in drug prices, which affect the steady supply of drugs. A "basic drug" system was introduced to rescue these drugs by eliminating gaps in drug prices among preparations with the same constituents. "Type 1" hypotonic infusion fluids, which are potassium-free and commonly used to treat dehydration, meet the definition of a "basic drug" in Japan, and there are no drug price gaps. However, there is a lack of information on the physicochemical properties of "type 1" hypotonic infusion fluids, making it difficult to identify differences among them. Extracellular fluid-replacement solutions and "type 3" hypotonic infusion fluids have different pH and titratable acidity. Here, we measured the pH, titratable acidity, and osmolality of six different "type 1" hypotonic infusion fluids and compared the results with respect to risk avoidance considering metabolic acidosis, changes upon mixing, and vascular pain. There was a significant difference, or trend toward significance, in titratable acidity, which is a risk factor for metabolic acidosis in patients with impaired renal function, and pH, which is a risk factor for change upon mixing, among all combinations except one of the infusion fluids. Thus, the selection of "type 1" hypotonic infusion fluids for children with immature renal function, elderly patients with impaired renal function, and patients with unknown pathophysiology, considering titratable acidity and pH, is an effective strategy for risk avoidance.

A nonlinear and time-dependent leak current in the presence of calcium fluoride patch-clamp seal enhancer

Automated patch-clamp platforms are widely used and vital tools in both academia and industry to enable high-throughput studies such as drug screening. A leak current to ground occurs whenever the seal between a pipette and cell (or internal solution and cell in high-throughput machines) is not perfectly insulated from the bath (extracellular) solution. Over 1 GΩ seal resistance between pipette and bath solutions is commonly used as a quality standard for manual patch work. With automated platforms it can be difficult to obtain such a high seal resistance between the intra- and extra-cellular solutions. One suggested method to alleviate this problem is using an F − containing internal solution together with a Ca 2+ containing external solution — so that a CaF 2 crystal forms when the two solutions meet which ‘plugs the holes’ to enhance the seal resistance. However, we observed an unexpected nonlinear-in-voltage and time-dependent current using these solutions on an automated patch-clamp platform. We performed manual patch-clamp experiments with the automated patch-clamp solutions, but no biological cell, and observed the same nonlinear time-dependent leak current. The current could be completely removed by washing out F − ions to leave a conventional leak current that was linear and not time-dependent. We therefore conclude fluoride ions interacting with the CaF 2 crystal are the origin of the nonlinear time-dependent leak current. The consequences of such a nonlinear and time-dependent leak current polluting measurements should be considered carefully if it cannot be isolated and subtracted.

Effective Perturbations on the Amplitude and Hysteresis of Erg-Mediated Potassium Current Caused by 1-Octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6(undecyloxy)hexyl]amino]-octanoate (SM-102), a Cationic Lipid

SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate) is an amino cationic lipid that has been tailored for the formation of lipid nanoparticles and it is one of the essential ingredients present in the ModernaTM COVID-19 vaccine. However, to what extent it may modify varying types of plasmalemmal ionic currents remains largely uncertain. In this study, we investigate the effects of SM-102 on ionic currents either in two types of endocrine cells (e.g., rat pituitary tumor (GH3) cells and mouse Leydig tumor (MA-10) cells) or in microglial (BV2) cells. Hyperpolarization-activated K+ currents in these cells bathed in high-K+, Ca2+-free extracellular solution were examined to assess the effects of SM-102 on the amplitude and hysteresis of the erg-mediated K+ current (IK(erg)). The SM-102 addition was effective at blocking IK(erg) in a concentration-dependent fashion with a half-maximal concentration (IC50) of 108 μM, a value which is similar to the KD value (i.e., 134 μM) required for its accentuation of deactivation time constant of the current. The hysteretic strength of IK(erg) in response to the long-lasting isosceles-triangular ramp pulse was effectively decreased in the presence of SM-102. Cell exposure to TurboFectinTM 8.0 (0.1%, v/v), a transfection reagent, was able to inhibit hyperpolarization-activated IK(erg) effectively with an increase in the deactivation time course of the current. Additionally, in GH3 cells dialyzed with spermine (30 μM), the IK(erg) amplitude progressively decreased; moreover, a further bath application of SM-102 (100 μM) or TurboFectin (0.1%) diminished the current magnitude further. In MA-10 Leydig cells, the IK(erg) was also blocked by the presence of SM-102 or TurboFectin. The IC50 value for SM-102-induced inhibition of IK(erg) in MA-10 cells was 98 μM. In BV2 microglial cells, the amplitude of the inwardly rectifying K+ current was inhibited by SM-102. Taken together, the presence of SM-102 concentration-dependently inhibited IK(erg) in endocrine cells (e.g., GH3 or MA-10 cells), and such action may contribute to their functional activities, assuming that similar in vivo findings exist.

Theoretical Estimation of Optimal Linear Cooling Rate for PK-15 Cell Suspension

Preservation of cells during crystallization of the cell suspension is influenced by two types of damaging factors. The first type of cryoinjury occurs during the crystallization of the extracellular environment and is caused by dehydration of cells, increasing the concentration and ionic strength of extracellular and intracellular solutions. As the cooling rate rises, the damage rate of the first type decreases as a result of the reduced time of action of damaging factors. The second type of cryoinjury is intracellular crystallization, the probability of which enhances at high cooling rates, is considered the most destructive to cells. The optimal linear cooling rate for PK-15 cells is determined using a physico-mathematical model, which describes the probability of cryoinjury of cells in the linear freezing mode and is based on the two-factor theory of cryoinjury, thermodynamic theory of homogeneous crystallization and general theory of activation-type processes. The findings have shown that within the range of cooling rates < 0.5 °C/min the cryoinjury of PK-15 cells occurs mainly due to the effects of the solution, and at cooling rates > 2.5 °C/min this was mainly resulted from an intracellular crystallization. The dependence of the percentage of damaged cells on the cooling rate has a relatively wide minimum within the range of cooling rates of 0.5 °C/min… 2.5 °C/min.

Dopaminergic Inhibition of Na+ Currents in Vestibular Inner Ear Afferents

Inner ear hair cells form synapses with afferent terminals and afferent neurons carry signals as action potentials to the central nervous system. Efferent neurons have their origins in the brainstem and some make synaptic contact with afferent dendrites beneath hair cells. Several neurotransmitters have been identified that may be released from efferent terminals to modulate afferent activity. Dopamine is a candidate efferent neurotransmitter in both the vestibular and auditory systems. Within the cochlea, activation of dopamine receptors may reduce excitotoxicity at the inner hair cell synapse via a direct effect of dopamine on afferent terminals. Here we investigated the effect of dopamine on sodium currents in acutely dissociated vestibular afferent calyces to determine if dopaminergic signaling could also modulate vestibular responses. Calyx terminals were isolated along with their accompanying type I hair cells from the cristae of gerbils (P15-33) and whole cell patch clamp recordings performed. Large transient sodium currents were present in all isolated calyces; compared to data from crista slices, resurgent Na+ currents were rare. Perfusion of dopamine (100 μM) in the extracellular solution significantly reduced peak transient Na+ currents by approximately 20% of control. A decrease in Na+ current amplitude was also seen with extracellular application of the D2 dopamine receptor agonist quinpirole, whereas the D2 receptor antagonist eticlopride largely abolished the response to dopamine. Inclusion of the phosphatase inhibitor okadaic acid in the patch electrode solution occluded the response to dopamine. The reduction in calyx sodium current in response to dopamine suggests efferent signaling through D2 dopaminergic receptors may occur via common mechanisms to decrease excitability in inner ear afferents.

Enhancing the degradation of Aflatoxin B1 by co-cultivation of two fungi strains with the improved production of detoxifying enzymes

After the co-culture of Aspergillus niger and Pleurotus ostreatus, the obtained extracellular crude enzymes solution was employed to aflatoxin B1 (AFB1) degradation. The maximum AFB1 degradation with co-culture reached 93.4%, which increased by 65.9% and 37.6%, respectively, compared with those of the mono-culture of Pleurotus ostreatus and Aspergillus niger. The molecular weight of the key detoxifying enzymes isolated by ultrafiltration was 58 and 63 kDa by SDS-PAGE analysis. The purified detoxifying enzymes had a high detoxification effect on AFB1 with the degradation rate of 94.7%. It was found that the co-culture of Pleurotus ostreatus and Aspergillus niger promoted the production of 58 and 63 kDa detoxifying enzymes to enhance the AFB1 degradation. The chemical structure of major degradation products of AFB1 by the mixed cultures were preliminarily identified by LC-Triple TOF MS. Two pathways of AFB1 degradation were inferred with the high potential of fungal co-cultivations for AFB1 detoxification applications.

The role of the SARS-CoV-2 envelope protein as a pH-dependent cation channel.

The role of the SARS-CoV-2 envelope protein as a pH-dependent cation channel.

Molecular dynamics simulation of human urea transporter B

ABSTRACT We applied a new large two membrane model of human urea transporter B (UT-B) with urea concentration gradient between intracellular and extracellular solution to explore the structure and dynamics of urea permeation. The first solved human UT-B X-ray crystal structure (PDB 6QD5) was studied using atomistic force field based large scale molecular dynamics (MD) simulations. The urea binding sites in UT-B were identified by the combination of first principles molecular dynamics docking and hybrid steered molecular dynamics (hSMD). In the simulations, urea molecules bound to the two binding sites although urea molecules did not go through the channels. The potential of mean force (PMF) of urea along the channel was calculated by hSMD to investigate the selectivity of the urea permeation across the channel pore. The simulations provide an opportunity to explore the binding sites spontaneously to help understand the urea permeation gating and transportation mechanism. A clearer analysis of the selectivity filter is shown in more detail. We also furthered the research about small organic molecules in the specific environment, such as channels in this work.

Does the solution used for cold static storage of hearts impact on heart transplant survival?

A best evidence topic in cardiac surgery was written according to a structured protocol. The question addressed was 'For the [cold static storage of donor hearts] does the [cardiac preservation solution] impact on [heart transplant survival]?'. Altogether 182 papers were found using the reported search, of which 9 represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. A wide variety of both custom and standardized cardiac preservation solutions are used for the procurement and cold static storage of donor hearts. In heart transplants with shorter cold ischaemic times, survival outcomes appear similar across many of the common cardiac preservation solutions in the long term. However, unspecified cardioplegia (with supraphysiological potassium concentration) is not supported as the final storage solution as it was associated with worse outcomes overall. In the short term, intracellular solutions (<70 mEq/l sodium; including University of Wisconsin solution) may also have better survival outcomes than extracellular solutions. Where extended ischaemic times (>4 h) are necessary or high-risk grafts obtained, the evidence supports University of Wisconsin as the preferred solution. We concluded that choice of cardiac preservation solution could impact both survival and secondary outcomes.

Monovalent: Divalent Anion Selectivity in the CFTR Channel Pore.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel shows only weak selectivity between different small monovalent anions, however, little is known about its ability to discriminate between monovalent and divalent anions. The present study uses patch clamp recording to investigate the interaction between the small divalent anions S2O32- and SO42- and wild-type and pore-mutant forms of human CFTR. Binding of these anions to wild-type CFTR appears weak; at 10 mM, intracellular S2O32- and SO42- blocked <20 and <5% of macroscopic Cl- current respectively, while these same concentrations had no discernible blocking effect when present in the extracellular solution. However, introduction of additional positive charge into the inner vestibule of the pore (in I344K and S1141K mutant channels) drastically strengthened block by intracellular (but not extracellular) S2O32- and SO42-. Block of these mutant channels was highly voltage-dependent; at very negative membrane potentials, apparent binding affinities were ~100 µM for S2O32- and <1 mM for SO42-. Permeability of S2O32- and SO42- was too small to be quantified in wild-type CFTR, but was <1% of Cl- permeability. Mutants that strengthened divalent binding (I344K, S1141K), as well as the selectivity-altering mutant F337A, also showed immeasurably low S2O32- and SO42- permeabilities. Overall CFTR selects well for monovalent over divalent anions, both in terms of binding and permeability. The number or density of fixed positive charges in the pore appears well optimized to disfavour binding of divalent anions, which may be an important facet of the monovalent Cl- permeation mechanism.

Angiotensin II Elicits Intracellular Ca 2+ Responses in Rat Cortical Collecting Ducts

Regulation of sodium transport in the cortical collecting duct (CCD) is pivotal for the long-term blood pressure control and excessive Na+ retention in this nephron segment is a known pathogenic determinant of hypertension. Sodium reabsorption in the CCD cells can be stimulated by volume-regulatory hormones, such as angiotensin II (AngII). AngII reportedly binds to AT1 receptors and increases the activity of the epithelial sodium channel (ENaC) via generation of reactive oxygen species (ROS). Yet, our understanding of the exact molecular determinants mediating AngII-induced intracellular signaling in the CCD cells remains limited. Numerous studies show that AngII induces intracellular Ca2+ mobilization in the vasculature, cardiomyocytes and glomerular podocytes. Here, we used ratiometric Fura-2 Ca2+-imaging to test if AngII employs Ca2+ as a second messenger in the CCDs freshly isolated from male and female Sprague-Dawley (SD) rats. We found that application of 500 nM of AngII to a split-opened CCD results in a transient elevation of [Ca2+]i in the CCD cells. The amplitude of the observed [Ca2+]i increase is about 150 nM and is not significantly different in the CCDs isolated from male and female rats. AngII-dependent [Ca2+]i signal is not affected by removal of Ca2+ from the extracellular bathing solution, but is practically abolished, when the tubules are pre-incubated in 3 μM thapsigargin, indicative of Ca2+ release from the intracellular stores. Inhibition of PLC with 10 μM U73122 markedly diminishes AngII-induced [Ca2+]i transient. Chronic AngII infusion moderately impairs [Ca2+]i responses to AngII in the CCDs isolated from male rats by approximately 30%. In contrast, AngII-induced [Ca2+]i elevation is attenuated by 70% in the CCDs from AngII-infused females. We have previously reported a stronger antihypertensive effect of aldosterone antagonism in AngII-infused females, when compared to males. Here, we found that systemic administration of eplerenone prevents the decline of AngII-stimulated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension, but not in males. Overall, our data demonstrate that AngII triggers Ca2+ release from the intracellular stores of the CCD cells in a PLC-dependent manner. This AngII-activated [Ca2+]i mobilization is impaired in AngII-infused SD rats of both sexes, but the attenuation is remarkably more pronounced in females. Systemic aldosterone antagonism with eplerenone restores the AngII-mediated [Ca2+]i response in the CCDs from female rats with AngII-dependent hypertension. Future studies are granted to investigate the interplay between AngII-dependent [Ca2+]i signal and ROS generation in the CCD cells.

Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1.

Pain at the injection site is a common complaint of patients receiving therapeutic formulations containing citric acid. Despite the widely acknowledged role of acid-sensing ion channels (ASICs) in acid-related perception, the specific ASIC subtype mediating pain caused by subcutaneous acid injection and the mechanism by which citrate affects this process are less clear. Here, male mice subjected to intraplantar acid injection responded by executing a withdrawal reflex, and this response was abolished by ASIC1 but not ASIC2 knockout. Although intraplantar injection of neutral citrate solution did not produce this response, intraplantar injection of acidic citrate solution produced a withdrawal reflex greater than that produced by acidity alone. Consistent with the behavioral data, neutral citrate failed to produce an electrophysiological response in HEK293 cells, which express ASIC1, but acidic citrate produced a whole-cell inward current greater than that produced by acidity alone. Saturating the intracellular solution with citrate had no effect on the potentiating effect of extracellular citrate, suggesting that citrate acted extracellularly to potentiate ASIC1. Moreover, exposure to citrate immediately before acid stimulation failed to potentiate ASIC1 currents, which ruled out the involvement of a metabotropic receptor gated by a citrate metabolite. Finally, removal of calcium ions from the extracellular solution mimicked the potentiating effect of citrate and prevented citrate from further potentiating ASIC1. Our data demonstrate that ASIC1 is necessary for the nociceptive response caused by subcutaneous acid infusion and that neutral citrate, despite not inducing ASIC1 currents or nociceptive behavior on its own, potentiates acid nociception by removing the inhibitory effect of extracellular calcium ions on ASIC1.SIGNIFICANCE STATEMENT Citric acid is a common ingredient used in pharmaceutical formulations. Despite the widespread clinical use of these formulations, it remains unclear how citric acid causes pain when injected into patients. We identified ASIC1 as the key receptor used to detect injection-site pain caused by acid, and we showed that neutral citrate does not stimulate ASIC1; instead, citrate substantially potentiates ASIC1 activation when injected simultaneously with acid. In addition, we demonstrated that citrate potentiates ASIC1 by removing the inhibitory action of calcium on the extracellular side of the receptor. Given that injection-site pain is the primary complaint of patients receiving citrate-containing medical products, our data provide mechanistic insight into a common medical complaint and suggest a means of avoiding injection pain.

Bicarbonate ion transport by the electrogenic Na+ /HCO3- cotransporter, NBCe1, is required for normal electrical slow-wave activity in mouse small intestine.

Normal gastrointestinal motility depends on electrical slow-wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3- in extracellular solutions used to record slow waves indicates a role for HCO3- transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+ /HCO3- cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC-MY) that generate slow waves. This study aimed to determine how extracellular HCO3- concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects. Immunohistochemistry and sharp electrode electrical recordings were used. The NBCe1 immunoreactivity was localized to ICC-MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3- from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow-wave amplitude and frequency. In 100mM HCO3- , the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+ , an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3- . There were no additional changes to electrical activity when HCO3- was removed from Li+ containing solutions. The Na+ /HCO3- cotransport inhibitor, S-0859 (30µM) significantly reduced the effect of removing HCO3- on electrical activity. These studies demonstrate a major role for Na+ /HCO3- cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract.