Danny Jans

Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6)

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin‐luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing‐type chamber. We developed a LL pulse protocol to determine the rate of ATP release (RATP) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure RATP as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 μl of A6 cells released ATP at a rate of 66 ± 8 fmol min−1. A sudden reduction of the basolateral osmolality from 260 to 140 mosmol (kg H2O)−1 elevated RATP rapidly to a peak value of 1.89 ± 0.11 pmol min−1 (RATPpeak) followed by a plateau phase reaching 0.51 ± 0.07 pmol min−1 (RATPplat). Both RATPpeak and RATPplat values increased with the degree of dilution. The magnitude of RATPplat remained constant as long as the hyposmolality was maintained. Similarly, a steady ATP release of 0.78 ± 0.08 pmol min−1 was recorded after gradual dilution of the basolateral osmolality to 140 mosmol (kg H2O)−1. This RATP value, induced in the absence of cell swelling, is comparable to RATPplat. Therefore, the steady ATP release is unrelated to membrane stretching, but possibly caused by the reduction of intracellular ionic strength during cell volume regulation. Independent determinations of dose‐response curves for peak [Ca2+]i increase in response to exogenous ATP and basolateral hyposmolality demonstrated that the exogenous ATP concentration, required to mimic the osmotic reduction, was linearly correlated with RATPpeak. The link between the ATP release and the fast [Ca2+]i transient was also demonstrated by the depression of both phenomena by Cl− removal from the basolateral perfusate. The data are consistent with the notion that during hypotonicity, basolateral ATP release activates purinergic receptors, which underlies the suramin‐sensitive rise of [Ca2+]i during the hyposmotic shock.

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.

Transepithelial capacitance decrease reveals closure of lateral interspace in A6 epithelia.

Abstract A sine wave method was used to measure transepithelial capacitance (CT) at 4.1 kHz (CHFT ). Model calculations show that CHFT reflects the equivalent capacitance of the series arrangement of apical and basolateral membrane capacitance. Cell swelling induced by reducing the basolateral osmolality from 260 to 140 mosmol/kg H2O (NaCl or sucrose removal) transiently decreased CHFT . The decrease in CHFT (ΔCHFT ) reached its maximum 30 s after the onset of cell swelling and a complete recovery of CHFT was attained within 3–4 min. ΔCHFT could be diminished by manoeuvres that reduced the rate or amplitude of cell swelling, i.e. lowering the temperature or treatment with low concentrations of glutaraldehyde (0.025%). ΔCHFT increased with the magnitude of the osmotic perturbation but saturated at large volume expansions. ΔCHFT increased with culture time. Electron micrographs showed a clear correlation between time course of CHFT changes and the closure of the lateral interspace (LIS). A striking correlation between the occurrence of CHFT recovery and the ability of the cells to develop a regulatory volume decrease (RVD) was found: Gd3+ (0.5 mM) inhibited both phenomena. The frequency dependence of CT was obtained from impedance spectra recorded over the range of 4 Hz to 22 kHz. These data agree with model calculations in which the contribution of the access resistance to the lateral membrane was included. All observations are consistent with the idea that ΔCHFT originates from the closure of the LIS during cell swelling. The latter phenomenon increases the access resistance to the lateral membrane, which results in a marked reduction of the basolateral membrane area detected at high frequencies with capacitance measurements.

Measurement of rapid changes in cell volume by forward light scattering.

Light scattering is an empirical technique employed to measure rapid changes in cell volume. This study describes a new configuration for the method of light scattering and its corroboration by measurements of cell height (as a measure of cell volume). Corneal endothelial cells cultured on glass cover-slips were mounted in a perfusion chamber on the stage of an inverted microscope. A beam of light was focused on the cells from above the stage at an angle of 40° to the plane of the stage. The scattered light intensity (SLI), captured by the objective and referred to as forward light scatter (FLS), increased and decreased in response to hyposmotic and hyperosmotic shocks, respectively. The rapid increase and decrease in SLI corresponded to cell swelling and shrinkage, respectively. Subsequently, SLI decreased and increased as expected for a regulatory volume decrease (RVD) and increase (RVI), respectively. These data are in agreement with measurements of cell height, demonstrating that the method of light scatter in FLS mode is useful for monitoring rapid changes in cell volume of cultured cells. Changes in SLI caused by gramicidin were consistent with cell volume changes induced by equilibration of NaCl and KCl concentrations across the cell membrane. Similarly, an additional decrease in SLI was recorded during RVD upon increasing K+ conductance by valinomycin. Decreasing K+ conductance of the cell membrane with Ba2+ changed the time course of SLI consistent with the effect of the K+ channel blocker on RVD. Bumetanide and dihydro-ouabain inhibited increases in SLI during RVI. In conclusion, FLS is a valid method for qualitative analysis of cell volume changes with a high time resolution.

Mg2+-sensitive non-capacitative basolateral Ca2+ entry secondary to cell swelling in the polarized renal A6 epithelium

Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (CT) that is inhibited by extracellular Mg2+. Elevation of free cytosolic [Ca2+] ([Ca2+]i) is known to increase CT. Therefore, we examined [Ca2+]i dynamics and their sensitivity to extracellular Mg2+ during hyposmotic conditions. Fura‐2‐loaded A6 monolayers, cultured on permeable supports were subjected to a sudden reduction in osmolality at both the basolateral and apical membranes from 260 to 140 mosmol (kg H2O)−1. Reduction of apical osmolality alone did not affect [Ca2+]i. In the absence of extracellular Mg2+, the hyposmotic shock induced a biphasic rise in [Ca2+]i. The first phase peaked within 40 s and [Ca2+]i increased from 245 ± 12 to 606 ± 24 nm. This phase was unaffected by removal of extracellular Ca2+, but was abolished by activating P2Y receptors with basolateral ATP or by exposing the cells to the phospholipase C (PLC) inhibitor U73122 prior to the osmotic shock. Suramin also severely attenuated this first phase, suggesting that the first phase of the [Ca2+]i rise followed swelling‐induced ATP release. The PLC inhibitor, the ATP treatment or suramin did not affect a second rise of [Ca2+]i to a maximum of 628 ± 31 nm. The second phase depended on Ca2+ in the basolateral perfusate and was largely suppressed by 2 mm basolateral Mg2+. Acute exposure of the basolateral membrane to Mg2+ during the upstroke of the second phase caused a rapid decline in [Ca2+]i. Basolateral Mg2+ inhibited Ca2+ entry in a dose‐dependent manner with an inhibition constant (Ki) of 0.60 mm. These results show that polarized A6 epithelia respond to hyposmotic shock by Ca2+ release from inositol trisphosphate‐sensitive stores, followed by basolateral Ca2+ influx through a Mg2+‐sensitive pathway. The second phase of the [Ca2+]i response is independent of the initial intracellular Ca2+ release and therefore constitutes non‐capacitative Ca2+ entry.

Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations.

Effects of basolateral monovalent cation replacements (Na+ by Li+, K+, Cs+, methylammonium, and guanidinium) on permeability to86Rb of volume-sensitive cation channels (VSCC) in the basolateral membrane and on regulatory volume decrease (RVD), elicited by a hyposmotic shock, were studied in A6 epithelia in the absence of apical Na+ uptake. A complete and quick RVD occurred only when the cells were perfused with Na+ or Li+ saline. With both cations, hypotonicity increased basolateral86Rb release ([Formula: see text]), which reached a maximum after 15 min and declined back to control level. When the major cation was K+, Cs+, methylammonium, or guanidinium, the RVD was abolished. Methylammonium induced a biphasic time course of cell thickness (Tc), with an initial decline of Tc followed by a gradual increase. With K+, Cs+, or guanidinium, Tc increased monotonously after the rapid initial rise evoked by the hypotonic challenge. In the presence of K+, Cs+, or methylammonium,[Formula: see text] remained high during most of the hypotonic period, whereas with guanidinium blockage of[Formula: see text] was initiated after 6 min of hypotonicity, suggesting an intracellular location of the site of action. With all cations, 0.5 mM basolateral Gd3+ completely blocked RVD and fully abolished the [Formula: see text] increase induced by the hypotonic shock. The lanthanide also blocked the additional volume increase induced by Cs+, K+, guanidinium, or methylammonium. When pH was lowered from 7.4 to 6.0, RVD and[Formula: see text] were markedly inhibited. This study demonstrates that the VSCCs in the basolateral membrane of A6 cells are permeable to K+, Rb+, Cs+, methylammonium, and guanidinium, whereas a marked inhibitory effect is exerted by Gd3+, protons, and possibly intracellular guanidinium.

Local electrical stimulation of single adherent cells using three-dimensional electrode arrays with small interelectrode distances

In this paper, we describe the localized and selective electrical stimulation of single cells using a three-dimensional electrode array. The chip consisted of 84 nail-like electrodes with a stimulation surface of 0.8 um and interelectrode distances as small as 3 um. N2A cells were used to compare bipolar stimulation between one electrode in- and one outside the cell on the one hand, and two electrodes in the same cell on the other hand. Selective and localized stimulation of primary embryonic cardiomyocytes showed the possibility to use this chip with excitable cells. The response of the cells to applied electrical fields was monitored using calcium imaging whereas assessment of electroporation was determined following influx of propidium iodide. Arrays of these three-dimensional electrodes could eventually be used as a tool to selectively electroporate the membrane of single cells for genetic manipulation or to obtain electrical access to the inner compartment of the cell.

Local electrical stimulation of cultured embryonic cardiomyocytes with sub-micrometer nail structures

In this paper, we demonstrate the feasibility of selective extracellular electrical stimulation at the (sub)cellular level in dissociated cultured cells. Using a CMOS-compatible process, we have fabricated an electrode array with sub-micrometer nail probes. Due to their particular configuration, the nails are strongly engulfed by the cellular membrane. By measuring the calcium signals, we found that electrical stimulation via the micronails activates the cell locally, in a dose-dependent manner, with very low applied currents. The results suggest the applicability of the device in pharmacological or signal propagation studies.

Action potential-based MEA platform for in vitro screening of drug-induced cardiotoxicity using human iPSCs and rat neonatal myocytes

Drug-induced cardiotoxicity poses a negative impact on public health and drug development. Cardiac safety pharmacology issues urged for the preclinical assessment of drug-induced ventricular arrhythmia leading to the design of several in vitro electrophysiological screening assays. In general, patch clamp systems allow for intracellular recordings, while multi-electrode array (MEA) technology detect extracellular activity. Here, we demonstrate a complementary metal oxide semiconductor (CMOS)-based MEA system as a reliable platform for non-invasive, long-term intracellular recording of cardiac action potentials at high resolution. Quinidine (8 concentrations from 10-7 to 2.10-5M) and verapamil (7 concentrations from 10-11 to 10-5M) were tested for dose-dependent responses in a network of cardiomyocytes. Electrophysiological parameters, such as the action potential duration (APD), rates of depolarization and repolarization and beating frequency were assessed. In hiPSC, quinidine prolonged APD with EC50 of 2.2·10-6M. Further analysis indicated a multifactorial action potential prolongation by quinidine: (1) decreasing fast repolarization with IC50 of 1.1·10-6M; (2) reducing maximum upstroke velocity with IC50 of 2.6·10-6M; and (3) suppressing spontaneous activity with EC50 of 3.8·10-6M. In rat neonatal cardiomyocytes, verapamil blocked spontaneous activity with EC50 of 5.3·10-8M and prolonged the APD with EC50 of 2.5·10-8M. Verapamil reduced rates of fast depolarization and repolarization with IC50s of 1.8 and 2.2·10-7M, respectively. In conclusion, the proposed action potential-based MEA platform offers high quality and stable long-term recordings with high information content allowing to characterize multi-ion channel blocking drugs. We anticipate application of the system as a screening platform to efficiently and cost-effectively test drugs for cardiac safety.

Micro-sized syringes for single-cell fluidic access integrated on a micro-electrode array CMOS chip

Very-large scale integration and micro-machining have enabled the development of novel platforms for advanced and automated examination of cells and tissues in vitro. In this paper, we present a CMOS chip designed in a commercial 0.18 μm technology with integrated micro-syringes combined with micro-nail shaped electrodes and readout electronics. The micro-syringes could be individually addressed by a through-wafer micro-fluidic channel with an inner diameter of 1 μm. We demonstrated the functionality of the micro-fluidic access by diffusion of fluorescent species through the channels. Further, hippocampal neurons were cultured on top of an array of micro-syringes, and focused ion beam-scanning electron microscopy cross-sections revealed protrusion of the cells inside the channels, creating a strong interface between the membrane and the chip surface. This principle demonstrates a first step towards a novel type of automated in vitro platforms, allowing local delivery of substances to cells or advanced planar patch clamping.