T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.     

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock

Combined blockade of Na⁺/Ca²⁺ exchange, Ca²⁺ uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca²⁺ after 1-min action of glutamate (100 M) or K⁺ (50 mM). However, replacement of Ca²⁺ with Ba²⁺, which cannot be transported by Ca²⁺-ATPase, considerably delayed the decrease in intracellular Ba²⁺ after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca²⁺-ATPase in Ca²⁺ extrusion after the action of glutamate or K⁺.     

Fluorescence and Laser Photon Counting: Measurements of Epithelial [Ca2+]i or [Na+]i with Ciliary Beat Frequency

We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca²⁺]i) or sodium ion concentration [Na⁺]i with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light beating experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10^–6, 10^–5, and 10^–4} M nifedipine on [Ca²⁺]i together with the CBF as well as the cumulative responses of 10^–5, 10^–4, and 10^–3 M amiloride on [Na⁺]i together with the CBF were also determined. Nifedipine decreased [Ca²⁺]i but had no effect on CBF. Amiloride decreased [Na⁺]i and CBF. Stimulation of CBF corresponded with either an increase of [Na⁺]i or an increase of [Ca²⁺]i. Decreases of [Na⁺]i or substantial decreases of [Ca²⁺]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia. © 1998 Biomedical Engineering Society. PAC98: 8780+s, 8722-q, 4262Be     

Role of TRP channels and NCX in mediating hypoxia-induced [Ca(2+)](i) elevation in PC12 cells

Mammalian cells require a constant O2 supply to produce adequate energy, and sustained hypoxia can kill cells. Mammals therefore have evolved sophisticated mechanisms to allow their cells to adapt to hypoxia. In this study, we investigated the role of TRP channels and the Na+-Ca2+ exchanger (NCX) in mediating hypoxia-induced [Ca2+]i elevation in a model of the O2-sensing rat pheochromocytoma (PC12) cell line by using Ca2+ imaging and molecular biological approaches. Non-selective cation channels, such as TRPC1, 3 and 6, were found to be functionally expressed in PC12 cells. They mediated Ca2+ entry when cells were exposed to acute hypoxia (PO2 of 15 mmHg), in addition to Ca2+ entry via VGCCs. Blockage of TRPCs by 2APB and SKF96365 could significantly reduce hypoxia-mediated [Ca2+]i elevation. Suramin and U73122 attenuated the hypoxia-induced [Ca2+]i elevation, implying the involvement of the G-protein and PLC pathways in the hypoxic response. In addition to TRPCs and VGCCs, NCX also contributed to the hypoxia-induced [Ca2+]i elevation, and blockade of NCX by KBR7943 could significantly decrease the hypoxia-induced [Ca2+]i elevation. Our results suggest that the activation of TRP by hypoxia could lead to NCX reversal; furthermore, membrane depolarization and TRPCs may play a primary role in mediating the hypoxic response in PC12 cells.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Na+ entry and modulation of Na+/Ca2+ exchange as a key mechanism of TRPC signaling

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca²⁺ homeostasis. As permeation of Ca²⁺ through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca²⁺ entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na⁺ permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na⁺ entry may be converted into a distinct pattern of cellular Ca²⁺ signals by interaction with Na⁺/Ca²⁺ exchanger proteins. This review discusses current concepts regarding the link between Na⁺ entry through TRPC channels and cellular Ca²⁺ signaling.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca²⁺ ([Ca²⁺]_i). The increases of [Ca²⁺]_i evoked by TG was associated with significant changes of plasma membrane Ca²⁺ permeability, with [Ca²⁺]_i values following changes in extracellular [Ca²⁺]. Plasma membrane Ca²⁺ extrusion is activated rapidly as a consequence of the rise in [Ca²⁺]_i evoked by TG and the rate of extrusion is linearly dependent on [Ca²⁺]_i up to 1 μM Ca²⁺. In contrast, the activation of the Ca²⁺ entry pathway is delayed and the apparent rate of Ca²⁺ entry is independent of [Ca²⁺]_i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP₃), the increase of [Ca²⁺]_i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca²⁺]_i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca²⁺]_i increase (from 12.3±1.0 to 6.1±0.6 nM Ca²⁺/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca²⁺]_i. The amplitude of the [Ca²⁺]_i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La³⁺, which is known to inhibit the plasma membrane Ca²⁺-activated adenosine triphosphatase, induced a much higher peak of [Ca²⁺]_i. This increase was associated with an augmentation of the apparent rate of [Ca²⁺]_i increase (from 12.3±1.2 to 16.1±1.9 nM Ca²⁺/s). The inhibitory effect of caffeine, as well as the increase in [Ca²⁺]_i observed on caffeine removal was not affected by the presence of 1 mM La³⁺. These data indicate that an important component of the TG-evoked [Ca²⁺]_i increase is due to InsP₃-sensitive Ca²⁺ release which is probably mediated by the resting levels of InsP₃.     

Is there a Na+/Ca2+ exchanger in macrophages and in lymphocytes?

In two blood cell types, peritoneal murine macrophages and Jurkat cells (a human T cell line), we have examined whether a Na⁺/Ca²⁺ exchange was present and what could be its functional importance. In nonstimulated macrophages, the intracellular Ca²⁺ concentration, [Ca²⁺]_i, was unchanged when Li⁺ was substituted for external Na⁺. However, after stimulation by platelet-activating factor (PAF), the Ca²⁺ response was larger when the extracellular solution contained Li⁺ rather than Na⁺ ions. In stimulated macrophages, the rate of Ca²⁺ extrusion was smaller in a Li⁺-than in a Na⁺-containing medium. The net electrochemical gradient for ionic movements through the Na⁺/Ca²⁺ exchanger, during the course of the response of macrophages to PAF, was determined by combining the measurements of membrane potential (in patch-clamp), of [Ca²⁺]_i (with fura-2), and of the intracellular Na⁺ concentration (with sodium-binding benzofuran isophthalate). These results show that macrophages possess a Na⁺/Ca²⁺ exchange that only functions as a Ca²⁺ extruder, and this only when [Ca²⁺]_i has been increased, for instance following PAF stimulation. In T lymphocytes, before or after stimulation by an anti-CD3 antibody, no Na⁺/Ca²⁺ activity could be detected by measuring either [Ca²⁺]_i, or the rate of Ca²⁺ extrusion. Even if a Na⁺/ Ca²⁺ exchanger was present in these cells, its equilibrium potential would be such that it would not allow Ca²⁺ influx but only Ca²⁺ extrusion.     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

Increased Ca2+ channel currents in cerebellar Purkinje cells of the ataxic groggy rat

The ataxic groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats. Recent genetic analysis has identified the missense (M251K) mutation in the alpha(1) subunit of the Ca(V)2.1 (P/Q-type) voltage-dependent Ca(2+) channel gene (Cacna1a) of GRY rat. In this study, we found that high-voltage-activated (HVA) Ca(2+) channel currents in acutely dissociated Purkinje cells of GRY rats showed increased (not decreased) current density and depolarizing shift of the activation and inactivation curves compared with those of normal Wistar rats. In contrast low-voltage-activated (LVA) Ca(2+) channel currents of GRY rats showed no significant changes. These results suggest that functional alteration of Ca(2+) channel currents in cerebellar Purkinje cells of GRY rats is attributed to the change of HVA Ca(2+) channel currents, and that increased HVA Ca(2+) channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.     

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current,I AHP,by acetylcholine,but not by glutamate,in hippocampal neurons

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca²⁺-activated K⁺current,I _AHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) onI _AHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3,5-cyclic monophosphate (cAMP) analogue Rp-adenosine-3,5-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression ofI _AHP. In contrast, two specific peptide inhibitors of Ca²⁺/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD onI _AHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression ofI _AHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action onI _AHP.     

High-frequency fatigue of skeletal muscle: role of extracellular Ca2+

The present study evaluated whether Ca(2+) entry operates during fatigue of skeletal muscle. The involvement of different skeletal muscle membrane calcium channels and of the Na(+)/Ca(2+) exchanger (NCX) has been examined. The decline of force was analysed in vitro in mouse soleus and EDL muscles submitted to 60 and 110 Hz continuous stimulation, respectively. Stimulation with this high-frequency fatigue (HFF) protocol, in Ca(2+)-free conditions, caused in soleus muscle a dramatic increase of fatigue, while in the presence of high Ca(2+) fatigue was reduced. In EDL muscle, HFF was not affected by external Ca(2+) levels either way, suggesting that external Ca(2+) plays a general protective role only in soleus. Calciseptine, a specific antagonist of the cardiac isoform (alpha1C) of the dihydropyridine receptor, gadolinium, a blocker of both stretch-activated and store-operated Ca(2+) channels, as well as inhibitors of P2X receptors did not affect the development of HFF. Conversely, the Ca(2+) ionophore A23187 increased the protective action of extracellular Ca(2+). KB-R7943, a selective inhibitor of the reverse mode of NCX, produced an effect similar to that of Ca(2+)-free solution. These results indicate that a transmembrane Ca(2+) influx, mainly through NCX, may play a protective role during HFF development in soleus muscle.     

Potentiating and depressant effects of metabotropic glutamate receptor agonists on high-voltage-activated calcium currents in cultured retinal ganglion neurons from postnatal mice

This study was aimed at clarifying the role of metabotropic glutamate receptors (mGluRs) in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in postnatal mouse retinal ganglion neurons (RGNs). RGNs were maintained for 1–2 weeks in vitro by adding brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) to the culture medium. In order to select these cells for electrophysiological measurements, RGNs were vitally labelled with an antibody against Thy-1.2. Voltage-activated Ca²⁺ currents [I _Ca(V)] were recorded with patch electrodes in the wholecell configuration. It was found that racemic ±-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD) or its active enantiomer 1S,3R-ACPD rapidly and reversibly either enhanced or depressed I _Ca(V). Quisqualate (QA), l-2-amino-4-phosphonobutyrate (l-AP4) and the endogenous transmitter glutamate induced similar effects when ionotropic glutamate receptors were blocked with d-2-amino-5-phosphonovalerate (d-APV) and 6,7-dinitroquinoxaline-2, 3-dione (DNQX). - Conotoxin GVIA (-CgTx GVIA), but not nifedipine prevented modulation of I _Ca(V) by mGluR agonists. The depression of I _Ca(V) by t-ACPD was irreversible when cells were dialysed with guanosine-5-O-(3-thiotriphosphate) (GTP[-S]). Ratio measurements of fura-2 fluorescence in Thy-1+ cells showed that neither t-ACPD, QA nor l-AP4 affected [Ca²⁺]_i by liberation of Ca²⁺ from intracellular stores. Our results suggest that cultured RGNs express mGluRs. These receptors cannot induce Ca²⁺ release from intracellular stores but regulate [Ca²⁺]_i by a fast and reversible, G-protein-mediated action on a subpopulation of voltage-activated Ca²⁺ channels.     

3-Isobutyl-1-methylxanthine (IBMX) affects potassium permeability in rat sensory neurones via pathways that are sensitive and insensitive to [Ca2+]in

The effects of externally applied 3-isobutyl-1-methylxanthine (IBMX), in millimolar concentrations, on the membrane currents in dorsal root ganglia (DRG) neurones isolated from newborn rats were investigated using the amphotericin-based perforated patch-clamp technique. In some experiments, simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_in) were performed using fura-2 microfluorimetry. Applications of IBMX induced elevation of [Ca²⁺]_in resulting from Ca²⁺ release from caffeine-ryanodine-sensitive internal stores. In addition to Ca²⁺ release, IBMX produced a biphasic membrane current response comprised of an inward current transiently interrupted by outward current. The onset of the inward current slightly preceded the onset of the [Ca²⁺]_in transient, while the interrupting outward current developed synchronously with the [Ca²⁺]_in rise. The development of IBMX-induced outward current ultimately needed the [Ca²⁺]_in elevation. After the depletion of Ca²⁺ stores by IBMX or caffeine exposure, the subsequent IBMX challenge failed to produce both the [Ca²⁺]_in transient and outward membrane current, although the inward current remained unchanged. Both components of the IBMX-induced membrane current response had a reversal potential close to the K⁺ equilibrium potential and the IBMX-induced membrane current response disappeared while dialysing the cell interior with K⁺-free, Cs⁺-containing solutions suggesting their association with K⁺ channel activity. External administration of 10 mM tetraethylammonium chloride (TEA-Cl) evoked an inward current similar to that observed in response to IBMX; in the presence of TEA-Cl, IBMX application was almost unable to induce additional inward current. IBMX (5 mM) effectively (50%) inhibited K⁺ currents evoked by step depolarizations of membrane potential. We suggest that IBMX affects membrane permeability via activation of Ca²⁺-regulated K⁺ channels and direct inhibition of TEA-sensitive K⁺ channels.     

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K+-ATPase and the Na+/Ca2+-exchanger

Copper (Cu²⁺) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu²⁺ toxicity is still unclear. It has been suggested that the Na⁺/K⁺-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu²⁺ on the Na⁺ and Ca²⁺ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na⁺-binding benzofuran isophtalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu²⁺ on the Na⁺/K⁺-ATPase activity. Cu²⁺ and ouabain increase the cytoplasmic free Na⁺ concentration ([Na⁺]_i). Subsequent addition of Cu²⁺ after ouabain does not affect the rate of [Na⁺]_i increase. Cu²⁺ inhibits the Na⁺/K⁺-ATPase activity with an IC₅₀ of 51 M. The cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) remains unaffected for more than 10 min after the administration of Cu²⁺. Thereafter, [Ca²⁺]_i increases as a result of the Na⁺/Ca²⁺-exchanger operating in the reversed mode. The effects of Cu²⁺ on the Na⁺ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N,N,-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na⁺ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu²⁺ does not modify the general membrane permeability, but inhibits the Na⁺/K⁺-pump leading to an increase of [Na⁺]_i. As a consequence the operation mode of the Na⁺/Ca²⁺-exchanger reverses and [Ca²⁺]_i rises.The authors thank staff and coworkers of the Department of Neurology of the University Hospital Nijmegen, Nijmegen for their kind cooperation in obtaining muscle biopsies. Mr. Arie Oosterhof is gratefully acknowledged for culturing of the human muscle cells. The Prinses Beatrix Fonds and the Dutch-Chinese scientific exchange program contributed financial support for this study.     

Measurement of Ca2+-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ current (L-type) and inward current caused by Ca²⁺ release from the sarcoplasmic reticulum and carried by electrogenic Na⁺/Ca²⁺ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca²⁺-chelating compound. It has been shown previously that Ca²⁺-release-dependent inward current under these conditions is carried by electrogenic Na⁺/Ca²⁺ exchange. Furthermore, Ca²⁺-release-dependent inward current (the release signal) can be completely separated from triggering Ca²⁺ current if brief depolarizations for activating I _Ca are used. In the majority of cells that did not produce spontaneous Ca²⁺ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering I _Ca was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca²⁺-buffered cells produced spontaneous Ca²⁺ release, resulting in irregular transient inward currents at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca²⁺ release.Supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

Modulation of Ca2+ oscillation and apamin-sensitive,Ca2+-activated K+ current in rat gonadotropes

In rat pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) stimulates rhythmic release of Ca²⁺ from stores sensitive to inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃ ], which in turn induces an oscillatory activation of apamin-sensitive Ca²⁺-activated K⁺ current, I _K(Ca). Since GnRH also activates protein kinase C (PKC), we investigate the action of PKC while simultaneously measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) and I _K(Ca). Stimulation of PKC by application of phorbol 12-myristate 13-acetate (PMA) did not affect basal [Ca²⁺]_i. However, PMA or phorbol 12,13-dibutyrate (PdBu), but not the inactive 4-phorbol 12,13-didecanoate (4-PDD), reduced the frequency of GnRH-induced [Ca²⁺]_i oscillation and augmented the I _K(Ca) induced by any given level of [Ca²⁺]_i. The slowing of oscillations and the enhancement of I _K(Ca) were mimicked by synthetic diacylglycerol (1,2-dioctanoyl-sn-glycerol) and could be induced during ongoing oscillations that had been initiated irreversibly in cells loaded with guanosine 5-O-(3-thio-triphosphate) (GTP-[S]). In contrast, when oscillations were initiated by loading cells with Ins(1,4,5)P ₃, phorbol esters enhanced I _K(Ca) without affecting the frequency of oscillation. The protein kinase inhibitor, staurosporine, reduced I _K(Ca) without affecting [Ca²⁺]_i and partially reversed the phorbol-ester-induced slowing of oscillation. Therefore, activation of PKC has two rapid effects on gonadotropes. It slows [Ca²⁺]_i oscillations probably by actions on phospholipase C, and it enhances I _K(Ca) probably by a direct action on the channels.