RESUMO
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.
Assuntos
Potenciais de Ação/fisiologia , Antiarrítmicos/farmacologia , Cardiotoxinas/farmacologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Semicondutores , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Microeletrodos , Miócitos Cardíacos/efeitos dos fármacos , Quinidina/farmacologia , Ratos , Ratos WistarRESUMO
Embryonic cells are very robust in surviving dissection and culturing protocols and easily adapt to their in vitro environment. Despite these advantages, research in the olfactory field on cultured embryonic olfactory neurons is sparse. In this study, two primary rat olfactory explant cultures of different embryonic d (E17 and E20) were established, comprising epithelium and bulb. The functionality of these neurons was tested by measuring intracellular calcium responses to cAMP-inducing agents forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) with fluorescence microscopy. For E17, the responsive cell fraction increased over time, from an initial 3% at the 1 d in vitro (DIV) to a maximum of 19% at 11 DIV. The response of E20 neurons fluctuated over time around a more or less stable 13%. A logistic regression analysis indicated a significant difference between both embryonic d in the response to FSK + IBMX. In addition, of these functional neurons, 23.3% of E17 and 54.3% of E20 cultures were responsive to the odorant isoamyl acetate.
Assuntos
Neurônios Receptores Olfatórios/citologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Colforsina/farmacologia , Feminino , Microscopia de Fluorescência , Neurônios Receptores Olfatórios/efeitos dos fármacos , Neurônios Receptores Olfatórios/metabolismo , Gravidez , RatosRESUMO
The investigation of complex communication in cellular networks requires superior measurement tools than those available to date. Electrode arrays integrated onto silicon electronics are increasingly used to measure the electrical activity of cells in an automated and highly parallelized fashion, but they are restricted to recording extracellular potentials. Here, we report on an array of TiN electrodes built using standard silicon electronics for intracellular action potential recording. Intracellular access, possible at each of the 16 384 electrodes on the chip, was accomplished by local membrane electroporation using electrical stimulation with subcellular, micrometer-sized electrodes. Access to the cell interior was transient and could be tuned in duration by adapting the electroporation protocol. Intracellular sensing was found to be minimally invasive in the short and long-term, allowing consecutive intracellular recordings from the same cell over the course of days. Finally, we applied this method to investigate the effect of an ion channel blocker on cardiac electrical activity. This technique opens the door to massively parallel, long-term intracellular recording for fundamental electrophysiology and drug screening.
Assuntos
Técnicas Eletroquímicas/métodos , Animais , Contagem de Células , Linhagem Celular , Técnicas Eletroquímicas/instrumentação , Eletrodos , Camundongos , Compostos de Estanho/químicaRESUMO
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.
Assuntos
Eletrodos , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Animais , Células Cultivadas , Estimulação Elétrica , Feminino , Miócitos Cardíacos/citologia , Ratos , Ratos Wistar , SemicondutoresRESUMO
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.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Miniaturização/instrumentação , Óxidos/química , Semicondutores , Análise de Célula Única/instrumentação , Seringas , Animais , Células Cultivadas , Fluorescência , Camundongos , Microeletrodos , Microscopia Eletrônica de Varredura , Neurônios/citologiaRESUMO
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 microm(2) and interelectrode distances as small as 3 microm. 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.
Assuntos
Estimulação Elétrica/instrumentação , Eletroporação/instrumentação , Eletroporação/métodos , Animais , Cálcio/metabolismo , Técnicas de Cultura de Células/instrumentação , Eletroquímica/métodos , Eletrodos , Campos Eletromagnéticos , Desenho de Equipamento , Coração/embriologia , Humanos , Microeletrodos , Microscopia Eletrônica de Varredura , Ratos , Ratos WistarRESUMO
This paper describes the fabrication and in vitro testing of a device capable of chemically stimulating individual neurons. Electrophoretic actuation is used to locally induce the release of the neurotransmitter L-glutamate in a network of hippocampal neurons cultured on top of the device. Cell activation by the neurotransmitter is visualized using calcium imaging. Single-cell stimulation is demonstrated close to the release site after application of a voltage of 500 mV. Such a device could provide a useful tool in both basic and clinical neuroscience. In a broader context, this device can be used to locally release small amounts of chemical compounds in Lab-on-Chip and drug delivery applications.
Assuntos
Ácido Glutâmico/administração & dosagem , Neurotransmissores/metabolismo , Sinapses/metabolismo , Animais , Cálcio/química , Cálcio/metabolismo , Sistemas de Liberação de Medicamentos , Estimulação Elétrica , Eletrodos , Desenho de Equipamento , Fluorescência , Concentração de Íons de Hidrogênio , Modelos Teóricos , Desenho de Prótese , Ratos , Raios UltravioletaRESUMO
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.
Assuntos
Potenciais de Ação/fisiologia , Estimulação Elétrica/instrumentação , Células-Tronco Embrionárias/fisiologia , Microeletrodos , Miócitos Cardíacos/fisiologia , Processamento de Sinais Assistido por Computador/instrumentação , Animais , Células Cultivadas , Estimulação Elétrica/métodos , Células-Tronco Embrionárias/citologia , Desenho de Equipamento , Análise de Falha de Equipamento , Miócitos Cardíacos/citologia , Ratos , Ratos WistarRESUMO
In this study, we have investigated the dependence of Na+ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (Isc), transepithelial conductance (GT), and transepithelial capacitance (CT) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl-beta-cyclodextrin (mbetaCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mbetaCD treatment. However, apical mbetaCD application hampered the responses of Isc and GT to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in Isc demonstrated that apical mbetaCD treatment decreased the epithelial Na+ channel (ENaC) open probability (Po) in the steady state as well as after activation of Na+ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by CT measurements. Na+ transport activation by hypotonicity was abolished during basolateral mbetaCD treatment as a result of reduced Na+/K+ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na+/K+ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na+ transport by reducing ENaC Po.
Assuntos
Colesterol/metabolismo , Células Epiteliais/metabolismo , Rim/citologia , Lipídeos de Membrana/metabolismo , Sódio/metabolismo , Adenosina/farmacologia , Animais , Artefatos , Linhagem Celular , Polaridade Celular/fisiologia , Células Epiteliais/citologia , Canais Epiteliais de Sódio , Soluções Hipotônicas/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Pressão Osmótica , Ocitocina/farmacologia , Potássio/metabolismo , Canais de Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Xenopus laevis , beta-Ciclodextrinas/farmacologiaRESUMO
This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.
Assuntos
Brônquios/metabolismo , Tamanho Celular , Células Epiteliais/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Brônquios/citologia , Linhagem Celular Transformada , Tamanho Celular/efeitos dos fármacos , Cloretos/metabolismo , Colforsina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Humanos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Pressão Osmótica/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Radioisótopos de Rubídio/farmacologia , Tirfostinas/farmacologiaRESUMO
We investigated the involvement of intracellular and extracellular Ca2+ in the stimulation of Na+ transport during hyposmotic treatment of A6 renal epithelia. A sudden osmotic decrease elicits a biphasic stimulation of Na+ transport, recorded as increase in amiloride-sensitive short-circuit current (Isc) from 3.4 +/- 0.4 to 24.0 +/- 1.3 microA/cm2 (n = 6). Changes in intracellular Ca2+ concentration ([Ca2+]i) were prevented by blocking basolateral Ca2+ entry with Mg2+ and emptying the intracellular Ca2+ stores before the hyposmotic challenge. This treatment did not noticeably affect the hypotonicity-induced stimulation of Isc. However, the absence of extracellular Ca2+ severely attenuated Na+ transport stimulation by the hyposmotic shock, and Isc merely increased from 2.2 +/- 0.3 to 4.8 +/- 0.7 microA/cm2. Interestingly, several agonists of the Ca2+-sensing receptor, Mg2+ (2 mM), Gd3+ (0.1 mM), neomycin (0.1 mM), and spermine (1 mM) were able to substitute for extracellular Ca2+. When added to the basolateral solution, these agents restored the stimulatory effect of the hyposmotic solutions on Isc in the absence of extracellular Ca2+ to levels that were comparable to control conditions. None of the above-mentioned agonists induced a change in [Ca2+]i. Quinacrine, an inhibitor of PLA2, overruled the effect of the agonists on Na+ transport. In conclusion, we suggest that a Ca2+-sensing receptor in A6 epithelia mediates the stimulation of Na+ transport without the interference of changes in [Ca2+]i.
Assuntos
Cálcio/metabolismo , Sódio/metabolismo , Urotélio/metabolismo , Animais , Cálcio/farmacologia , Linhagem Celular , Condutividade Elétrica , Espaço Extracelular/metabolismo , Soluções Hipotônicas/farmacologia , Magnésio/metabolismo , Magnésio/farmacologia , Concentração Osmolar , Pressão Osmótica , Fosfolipases A/metabolismo , Fosfolipases A2 , Receptores de Detecção de Cálcio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Urotélio/citologia , Água/metabolismo , Xenopus laevisRESUMO
We developed a non-invasive technique for electrophysiological investigations of ion transport proteins endogenously or heterologously expressed in Xenopus laevis oocytes. We named this technique the transoocyte voltage clamp (TOVC). Whereas in the classical two-microelectrode voltage-clamp (TEVC) technique, the oocyte is impaled with two glass microelectrodes, we mount the egg in a modified Ussing chamber as used for transepithelial electrophysiological studies. The oocyte is introduced in a container that is positioned between the two chamber halves. Proper fixation of the oocyte in the aperture of the container is accomplished under a stereo binocular microscope and the electrical seal between the oocyte and the container is achieved with silicon grease. The new method allows measurement of transoocyte currents and conductances as well as the recording of membrane impedance and the fluctuation analysis of ion currents. We studied a K+ channel that resembles the inward rectifier K+ channel endogenously expressed in Xenopus laevis oocytes. K+ currents were obtained by exposing one side of the oocyte to K(+)-containing solutions and by the application of different voltages. Adding Cs+ and Ba2+ inhibited these currents. The analysis of the fluctuation in current demonstrated a Lorentzian component in the power density spectrum. With the transoocyte voltage clamped to zero, the corner frequency (fc) was 61+/-1.7 Hz. Imposed positive transoocyte potentials caused a downward shift of fc. These findings are consistent with previous data obtained using the TEVC technique, and extend the characterization of the channel with kinetic data obtained from noise analysis.
Assuntos
Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Animais , Eletrofisiologia , Feminino , Técnicas In Vitro , Ativação do Canal Iônico/fisiologia , Canais de Potássio/fisiologia , Xenopus laevisRESUMO
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 degrees 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.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/fisiologia , Luz , Animais , Bovinos , Tamanho Celular/fisiologia , Projetos de Pesquisa/normas , Espalhamento de RadiaçãoRESUMO
In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca(2+) concentration ([Ca(2+)](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 (R(ATP)) in the basolateral compartment. Therefore, the perfusion at the basolateral border was repetitively interrupted during brief periods (90 s) to measure R(ATP) as the slope of the initial rise in ATP content detected by LL luminescence. Under isosmotic conditions, 1 microl 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 H(2)O)(-1) elevated R(ATP) rapidly to a peak value of 1.89 +/- 0.11 pmol min(-1) (R(ATP)(peak)) followed by a plateau phase reaching 0.51 +/- 0.07 pmol min(-1) (R(ATP)(plat)). Both R(ATP)(peak) and R(ATP)(plat) values increased with the degree of dilution. The magnitude of R(ATP)(plat) 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 H(2)O)(-1). This R(ATP) value, induced in the absence of cell swelling, is comparable to R(ATP)(plat). 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 [Ca(2+)](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 R(ATP)(peak). The link between the ATP release and the fast [Ca(2+)](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 [Ca(2+)](i) during the hyposmotic shock.
Assuntos
Trifosfato de Adenosina/metabolismo , Células Epiteliais/metabolismo , Soluções Hipotônicas/farmacologia , Rim/metabolismo , Animais , Cálcio/metabolismo , Calibragem , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Rim/efeitos dos fármacos , Luciferases/metabolismo , Medições Luminescentes , Concentração Osmolar , Xenopus laevisRESUMO
Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (C(T)) that is inhibited by extracellular Mg(2+). Elevation of free cytosolic [Ca(2+)] ([Ca(2+)](i)) is known to increase C(T). Therefore, we examined [Ca(2+)](i) dynamics and their sensitivity to extracellular Mg(2+) 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 H(2)O)(-1). Reduction of apical osmolality alone did not affect [Ca(2+)](i). In the absence of extracellular Mg(2+), the hyposmotic shock induced a biphasic rise in [Ca(2+)](i). The first phase peaked within 40 s and [Ca(2+)](i) increased from 245 +/- 12 to 606 +/- 24 nM. This phase was unaffected by removal of extracellular Ca(2+), 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 [Ca(2+)](i) rise followed swelling-induced ATP release. The PLC inhibitor, the ATP treatment or suramin did not affect a second rise of [Ca(2+)](i) to a maximum of 628 +/- 31 nM. The second phase depended on Ca(2+) in the basolateral perfusate and was largely suppressed by 2 mM basolateral Mg(2+). Acute exposure of the basolateral membrane to Mg(2+) during the upstroke of the second phase caused a rapid decline in [Ca(2+)](i). Basolateral Mg(2+) inhibited Ca(2+) entry in a dose-dependent manner with an inhibition constant (K(i)) of 0.60 mM. These results show that polarized A6 epithelia respond to hyposmotic shock by Ca(2+) release from inositol trisphosphate-sensitive stores, followed by basolateral Ca(2+) influx through a Mg(2+)-sensitive pathway. The second phase of the [Ca(2+)](i) response is independent of the initial intracellular Ca(2+) release and therefore constitutes non-capacitative Ca(2+) entry.