Microvillar ion channels: cytoskeletal modulation of ion fluxes

The recently presented theory of microvillar Ca(2+)signaling [Lange, K. (1999) J. Cell. Physiol.180, 19-35], combined with Manning's theory of "condensed counterions" in linear polyelectrolytes [Manning, G. S. (1969). J. Chem. Phys.51, 924-931] and the finding of cable-like ion conductance in actin filaments [Lin, E. C. & Cantiello, H. F. (1993). Biophys. J.65, 1371-1378], allows a systematic interpretation of the role of the actin cytoskeleton in ion channel regulation.Ion conduction through actin filament bundles of microvilli exhibits unique nonlinear transmission properties some of which closely resemble that of electronic semiconductors: (1) bundles of microfilaments display significant resistance to cation conduction and (2) this resistance is decreased by supply of additional energy either as thermal, mechanical or electromagnetic field energy. Other transmission properties, however, are unique for ionic conduction in polyelectrolytes. (1) Current pulses injected into the filaments were transformed into oscillating currents or even into several discrete charge pulses closely resembling that of single-channel recordings. Discontinuous transmission is due to the existence of counterion clouds along the fixed anionic charge centers of the polymer, each acting as an "ionic capacitor". (2) The conductivity of linear polyelectrolytes strongly decreases with the charge number of the counterions; thus, Ca(2+)and Mg(2+)are effective modulator of charge transfer through linear polyelectrolytes. Field-dependent formation of divalent cation plugs on either side of the microvillar conduction line may generate the characteristic gating behavior of cation channels. (3) Mechanical movement of actin filament bundles, e.g. bending of hair cell microvilli, generates charge translocations along the filament structure (mechano-electrical coupling). (4) Energy of external fields, by inducing molecular dipoles within the polyelectrolyte matrix, can be transformed into mechanical movement of the system (electro-mechanical coupling). Because ionic transmission through linear polyelectrolytes is very slow compared with electronic conduction, only low-frequency electromagnetic fields can interact with the condensed counterion systems of linear polyelectrolytes.The delineated characteristics of microvillar ion conduction are strongly supported by the phenomenon of electro-mechanical coupling (reverse transduction) in microvilli of the audioreceptor (hair) cells and the recently reported dynamics of Ca(2+)signaling in microvilli of audio- and photoreceptor cells. Due to the cell-specific expression of different types and combinations of ion channels and transporters in the microvillar tip membrane of differentiated cells, the functional properties of this cell surface organelle are highly variable serving a multitude of different cellular functions including receptor-mediated effects such as Ca(2+)signaling, regulation of glucose and amino acid transport, as well as modulation of membrane potential. Even mechanical channel activation involved in cell volume regulation can be deduced from the systematic properties of the microvillar channel concept. In addition, the specific ion conduction properties of microfilaments combined with their proposed role in Ca(2+)signaling make microvilli the most likely cellular site for the interaction with external electric and magnetic fields.     

Role of microvillar cell surfaces in the regulation of glucose uptake and organization of energy metabolism

Experimental evidencesuggesting a type of glucose uptake regulation prevailing inresting and differentiated cells was surveyed. This type of regulationis characterized by transport-limited glucose metabolism and depends onsegregation of glucose transporters on microvilli of differentiated orresting cells. Earlier studies on glucose transport regulation and arecently presented general concept of influx regulation for ions andmetabolic substrates via microvillar structures provide the basicframework for this theory. According to this concept, glucose uptakevia transporters on microvilli is regulated by changes in thestructural organization of the microfilament bundle, which is acting asa diffusion barrier between the microvillar tip compartment and thecytoplasm. Both microvilli formation and the switch of glucosemetabolism from "metabolic regulation" to "transportlimitation" occur during differentiation. The formation ofmicrovillar cell surfaces creates the essential preconditions toestablish the characteristic functions of specialized tissue cellsincluding the coordination between glycolysis and oxidativephosphorylation, regulation of cellular functions by external signals,and Ca²⁺ signaling. The proposed concept integrates variousaspects of glucose uptake regulation into a ubiquitous cellularmechanism involved in regulation of transmembrane ion and substrate fluxes.

     

Activation of Calcium Signaling in Isolated Rat Hepatocytes Is Accompanied by Shape Changes of Microvilli

Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca²⁺signaling pathway, the ATP-dependent Ca²⁺store and the store-coupled Ca²⁺influx pathway, are both located in microvilli covering the surface of these cells. Microvilli-derived vesicles from both cell types exhibited anion and cation pathways which could be inhibited by anion and cation channel-specific inhibitors. These findings suggested that the microvillar tip compartment forms a space which is freely accessible for external Ca²⁺, ATP, and IP₃. The entry of Ca²⁺into the cytoplasm, however, is largely restricted by the microvillar core structure, the dense bundle of actin microfilaments acting as a diffusion barrier between the microvillar tip compartment and the cell body. Moreover, evidence has been presented that F-actin may function as ATP-dependent and IP₃-sensitive Ca²⁺store that can be emptied by profilin-induced depolymerization or reorganization [K. Lange and U. Brandt (1996)FEBS Lett.395, 137–142]. Here we demonstrate the tight connection between microvillar shape changes and the activation of the Ca²⁺signaling system in isolated rat hepatocytes. Using a combination of scanning electron microscopy (SEM) and fura-2 fluorescence technique, we confirmed a consequence of the “diffusion barrier” concept of Ca²⁺signaling: Irrespective of the type of the applied stimulus, activation of the Ca²⁺influx pathway is accompanied by changes in the structural organization of microvilli indicative of the loss of their diffusion barrier function. We further show that the cell surfaces of unstimulated hepatocytes isolated by either the collagenase or the EDTA perfusion technique are densely covered with microvilli predominantly of a short and slender type. Beside this rather uniformly shaped type of microvilli, a number of dilated surface protrusions were observed. Under these conditions the cells displayed the well known rather high basal [Ca²⁺]_iof 200–250 nMas repeatedly demonstrated for freshly isolated hepatocytes. However, addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), to the cell suspension immediately after its preparation reduced the basal cytoplasmic Ca²⁺level to about 100 nM.Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli. Activation of the Ca²⁺signaling pathway by vasopressin, as well as by the IP₃-independent acting Ca²⁺store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of [Ca²⁺]_iinduced by the effectors. Furthermore, the abundance of the large form of surface protrusions on isolated hepatocytes positively correlated with the size of a cellular Ca²⁺/Fura-2 compartment which is rapidly depleted from Ca²⁺by extracellular EGTA. These findings support the postulated localization of the store-coupled Ca²⁺influx pathway in microvilli of HIT cells also for hepatocytes and are in accord with the notion of a cytoskeletal diffusion barrier regulating the flux of external Ca²⁺via the microvillar tip region in the cytoplasm.     

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells

Background

It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca²⁺ mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca²⁺ release from Ca²⁺ stores in adrenal chromaffin cells.

Methods

We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca²⁺ in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2 h.

Results

Exposure to the magnetic field significantly reduced the increase in [Ca²⁺]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca²⁺ release by the exposure was unaffected.

Conclusions and general significance

These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca²⁺ release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca²⁺]i.     

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca²⁺, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca²⁺ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca²⁺ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X₃R > P2X_2b + X₄R > P2X_2bR > P2X_2a + X₄R > P2X₄R > P2X_2aR > P2X₇R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca²⁺ influx because of the coactivation of endogenously expressed Ca²⁺-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca²⁺ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca²⁺ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca²⁺ signals were not affected by voltage-gated Ca²⁺ influx and removal of extracellular sodium. Ca²⁺ signals reflected well the receptor-specific EC₅₀ values for ATP and the extracellular Zn²⁺ and pH sensitivities of P2XRs. These results indicate that intracellular Ca²⁺ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals     

Cellular mechanisms involved in CO(2) and acid signaling in chemosensitive neurons

An increase in CO₂/H⁺ is a major stimulus for increased ventilation and is sensed by specialized brain stem neurons called central chemosensitive neurons. These neurons appear to be spread among numerous brain stem regions, and neurons from different regions have different levels of chemosensitivity. Early studies implicated changes of pH as playing a role in chemosensitive signaling, most likely by inhibiting a K⁺ channel, depolarizing chemosensitive neurons, and thereby increasing their firing rate. Considerable progress has been made over the past decade in understanding the cellular mechanisms of chemosensitive signaling using reduced preparations. Recent evidence has pointed to an important role of changes of intracellular pH in the response of central chemosensitive neurons to increased CO₂/H⁺ levels. The signaling mechanisms for chemosensitivity may also involve changes of extracellular pH, intracellular Ca²⁺, gap junctions, oxidative stress, glial cells, bicarbonate, CO₂, and neurotransmitters. The normal target for these signals is generally believed to be a K⁺ channel, although it is likely that many K⁺ channels as well as Ca²⁺ channels are involved as targets of chemosensitive signals. The results of studies of cellular signaling in central chemosensitive neurons are compared with results in other CO₂- and/or H⁺-sensitive cells, including peripheral chemoreceptors (carotid body glomus cells), invertebrate central chemoreceptors, avian intrapulmonary chemoreceptors, acid-sensitive taste receptor cells on the tongue, and pain-sensitive nociceptors. A multiple factors model is proposed for central chemosensitive neurons in which multiple signals that affect multiple ion channel targets result in the final neuronal response to changes in CO₂/H⁺. hypercapnia; brain stem; ventilation; peripheral chemoreceptor; glia; gap junction; glomus; channel; calcium; potassium; carbonic anhydrase; taste receptor; nociception     

Involvement of stretch-activated cation channels in hypotonically induced insulin secretion in rat pancreatic beta-cells

In isolated rat pancreatic -cells, hypotonic stimulation elicited an increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) at 2.8 mM glucose. The hypotonically induced [Ca²⁺]_c elevation was significantly suppressed by nicardipine, a voltage-dependent Ca²⁺ channel blocker, and by Gd³⁺, amiloride, 2-aminoethoxydiphenylborate, and ruthenium red, all cation channel blockers. In contrast, the [Ca²⁺]_c elevation was not inhibited by suramin, a P₂ purinoceptor antagonist. Whole cell patch-clamp analyses showed that hypotonic stimulation induced membrane depolarization of -cells and produced outwardly rectifying cation currents; Gd³⁺ inhibited both responses. Hypotonic stimulation also increased insulin secretion from isolated rat islets, and Gd³⁺ significantly suppressed this secretion. Together, these results suggest that osmotic cell swelling activates cation channels in rat pancreatic -cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca²⁺ channels and thus elevating insulin secretion. calcium ion; swelling; patch-clamp; gadolinium     

Ca2+ sparks and T tubule reorganization in dedifferentiating adult mouse skeletal muscle fibers

Ca⁺ sparks are rare in healthy adult mammalian skeletal muscle but may appear when adult fiber integrity is compromised, and occur in embryonic muscle but decline as the animal develops. Here we used cultured adult mouse flexor digitorum brevis muscle fibers to monitor occurrence of Ca²⁺ sparks during maintenance of adult fiber morphology and during eventual fiber morphological dedifferentiation after various times in culture. Fibers cultured for up to 3 days retain normal morphology and striated appearance. Ca²⁺ sparks were rare in these fibers. At 5–7 days in culture, many of the original muscle fibers exhibit sprouting and loss of striations, as well as the occurrence of spontaneous Ca²⁺ sparks. The average rate of occurrence of Ca²⁺ sparks is >10-fold higher after 5–7 days in culture than in days 1–3. With the use of fibers cultured for 7 days, application of the Ca²⁺ channel blockers Co²⁺ or nifedipine almost completely suppressed the occurrence of Ca²⁺ sparks, as previously shown in embryonic fibers, suggesting that Ca²⁺ sparks may be generated by similar mechanisms in dedifferentiating cultured adult fibers and in embryonic fibers before final differentiation. The sarcomeric disruption observed under transmitted light microscopy in dedifferentiating fibers was accompanied by morphological changes in the transverse (T) tubular system, as observed by fluorescence confocal imaging of both an extracellular marker dye and membrane staining dyes. Changes in T tubule morphology coincided with the appearance of Ca²⁺ sparks, suggesting that Ca²⁺ sparks may either be a signal for, or the result of, disruption of DHPR-ryanodine receptor 1 coupling. calcium ion signaling; muscle remodeling; fluo 4; calcium ion imaging     

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor

Thecoupling mechanism between depletion of Ca²⁺ stores in theendoplasmic reticulum and plasma membrane store-operated ion channelsis fundamental to Ca²⁺ signaling in many cell types and hasyet to be completely elucidated. Using Ca²⁺release-activated Ca²⁺ (CRAC) channels in RBL-2H3 cells asa model system, we have shown that CRAC channels are maintained in theclosed state by an inhibitory factor rather than being opened by theinositol 1,4,5-trisphosphate receptor. This inhibitory role can befulfilled by the Drosophila protein INAD (inactivation-noafter potential D). The action of INAD requires Ca²⁺ andcan be reversed by a diffusible Ca²⁺ influx factor. Thusthe coupling between the depletion of Ca²⁺ stores and theactivation of CRAC channels may involve a mammalian homologue of INADand a low-molecular-weight, diffusible store-depletion signal.

     

Differential effect of Calcium and Magnesium on Mechanical Properties of Pea Stem Cell Walls

Replacement of calcium ion with magnesium ion in the cell wallof the pea epicotyl makes the wall more extensible. A possiblerole of this differential effect of Ca²⁺ and Mg^2– in regulatingcell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesiumion (Ca²⁺/Mg²⁺) in the walls decreased markedly in the orderof the first > the second > the third internodes of thepea epicotyl. The capacity of the walls for cation exchangeincreased in the same order, whereas the calcium-magnesium ionexchange selectivity of the walls was virtually constant. Ourresults indicate that the changes in the Ca²⁺/Mg²⁺ ratio amongthe internodes is not attributable to the ion exchange propertiesof the walk per se, but is due to other physiological conditionswhich regulate the activities of free calcium and magnesiumions in the environment with which the walls are in equilibrium. ¹ Present address: Wakayama Research Laboratories, Kao SoapCo., Ltd., Wakayama 640-91, Japan ² Present address: Foold Development Laboratories, Meiji SeikaKaisha Ltd., Kawasaki 210, Japan (Received May 1, 1981; Accepted August 26, 1981)     

Ionic mechanism for contractile response to hyposmotic challenge in canine basilar arteries

A hyposmotic challenge elicited contraction of isolated canine basilar arteries. The contractile response was nearly abolished by the removal of extracellular Ca²⁺ and by the voltage-dependent Ca²⁺ channel (VDCC) blocker nicardipine, but it was unaffected by thapsigargin, which depletes intracellular Ca²⁺ stores. The contraction was also inhibited by Gd³⁺ and ruthenium red, cation channel blockers, and Cl^– channel blockers DIDS and niflumic acid. The reduction of extracellular Cl^– concentrations enhanced the hypotonically induced contraction. Patch-clamp analysis showed that a hyposmotic challenge activated outwardly rectifying whole cell currents in isolated canine basilar artery myocytes. The reversal potential of the current was shifted toward negative potentials by reductions in intracellular Cl^– concentration, indicating that the currents were carried by Cl^–. Moreover, the currents were abolished by 10 mM BAPTA in the pipette solution and by the removal of extracellular Ca²⁺. Taken together, these results suggest that a hyposmotic challenge activates cation channels, which presumably cause Ca²⁺ influx, thereby activating Ca²⁺-activated Cl^– channels. The subsequent membrane depolarization is likely to increase Ca²⁺ influx through VDCC and elicit contraction. stretch-activated cation channels; Ca²⁺-activated Cl^– channels; voltage-dependent Ca²⁺ channels; large-conductance Ca²⁺-activated K⁺ channels; gadolinium     

Regulation of cAMP dynamics by Ca2+ and G protein-coupled receptors in the pancreatic -cell: a computational approach

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic β-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic β-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium (Ca²⁺) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic Ca²⁺ oscillations in the β-cell. Slow Ca²⁺ oscillations (<1 min^–1) produce low-frequency cAMP oscillations, and faster Ca²⁺ oscillations (>3–4 min^–1) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic Ca²⁺ oscillations. In contrast, observed antiphasic Ca²⁺ and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for Ca²⁺-dependent AC and PDE activation in coupling of Ca²⁺ and cAMP signals. Our results reveal important differences in the effects of glucose/TEA and GLP-1 on cAMP dynamics in MIN6 β-cells. adenylyl cyclase; calcium ion; glucagon-like peptide 1; modeling; oscillations     

An Excel-based model of Ca2+ diffusion and fura 2 measurements in a spherical cell

We wrote a program that runs as a Microsoft Excel spreadsheet to calculate the diffusion of Ca²⁺ in a spherical cell in the presence of a fixed Ca²⁺ buffer and two diffusible Ca²⁺ buffers, one of which is considered to be a fluorescent Ca²⁺ indicator. We modeled Ca²⁺ diffusion during and after Ca²⁺ influx across the plasma membrane with parameters chosen to approximate amphibian sympathetic neurons, mammalian adrenal chromaffin cells, and rat dorsal root ganglion neurons. In each of these cell types, the model predicts that spatially averaged intracellular Ca²⁺ activity ([Ca²⁺]_avg) rises to a high peak and starts to decline promptly on the termination of Ca²⁺ influx. We compared [Ca²⁺]_avg with predictions of ratiometric Ca²⁺ measurements analyzed in two ways. Method 1 sums the fluorescence at each of the two excitation or emission wavelengths over the N compartments of the model, calculates the ratio of the summed signals, and converts this ratio to Ca²⁺ ([Ca²⁺]_avg,M1). Method 2 sums the measured number of moles of Ca²⁺ in each of the N compartments and divides by the volume of the cell ([Ca²⁺]_avg,M2). [Ca²⁺]_avg,M1 peaks well after the termination of Ca²⁺ influx at a value substantially less than [Ca²⁺]_avg because the summed signals do not reflect the averaged free Ca²⁺ if the signals come from compartments containing gradients in free Ca²⁺ spanning nonlinear regions of the relationship between free Ca²⁺ and the fluorescence signals. In contrast, [Ca²⁺]_avg,M2 follows [Ca²⁺]_avg closely. intracellular calcium; kinetic model; diffusion coefficient; fura 2ff; furaptra     

The Ca(2+)-activated K(+) channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca²⁺ influx. KCa3.1 channels modulate Ca²⁺ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3 to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15–30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca²⁺ influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation. T cell activation; ion channels; membrane distribution     

Low-voltage triggering of Ca2+ release from the sarcoplasmic reticulum in cardiac muscle cells

This study investigated the interaction between L-type Ca²⁺ current (ICaL) and Ca²⁺ release from the sarcoplasmic reticulum (SRCR) in whole cell voltage-clamped guinea pig ventricular myocytes. Quasiphysiological cation solutions (Na_o⁺:K_I⁺) were used for most experiments. In control conditions, there was no obvious interaction between ICaL and SRCR. In isoproterenol, activation of ICaL from voltages between -70 and -50 mV reduced the amplitude and accelerated the decay of the current. Short (50 ms), small-amplitude voltage steps applied 60 or 510 ms before stimulating ICaL inhibited and facilitated the current, respectively. These changes were blocked by ryanodine. Low-voltage activated currents such as T-type Ca²⁺ current, TTX-sensitive ICa (ICaTTX), or slip mode Ca²⁺ conductance via INa⁺ were not responsible for low-voltage SRCR. However, L-type Ca²⁺ currents could be distinguished at voltages as negative as -45 mV. It is concluded that in the presence of isoproterenol, Ca²⁺ release from the SR at negative potentials is due to activation of L-type Ca²⁺ channels. heart; calcium current; low-voltage activation     

Interaction of reactive oxygen species with ion transport mechanisms

The use ofelectrophysiological and molecular biology techniques has shed light onreactive oxygen species (ROS)-induced impairment of surface andinternal membranes that control cellular signaling. These deleteriouseffects of ROS are due to their interaction with various ion transportproteins underlying the transmembrane signal transduction, namely,1) ion channels, such asCa²⁺ channels (includingvoltage-sensitive L-type Ca²⁺currents, dihydropyridine receptor voltage sensors, ryanodine receptorCa²⁺-release channels, andD-myo-inositol1,4,5-trisphosphate receptor Ca²⁺-release channels),K⁺ channels (such asCa²⁺-activatedK⁺ channels, inward and outwardK⁺ currents, and ATP-sensitiveK⁺ channels),Na⁺ channels, andCl channels;2) ion pumps, such as sarcoplasmicreticulum and sarcolemmal Ca²⁺pumps,Na⁺-K⁺-ATPase(Na⁺ pump), andH⁺-ATPase(H⁺ pump);3) ion exchangers such as theNa⁺/Ca²⁺exchanger andNa⁺/H⁺exchanger; and 4) ion cotransporterssuch asK⁺-Cl,Na⁺-K⁺-Cl,andP_i-Na⁺cotransporters. The mechanism of ROS-induced modificationsin ion transport pathways involves1) oxidation of sulfhydryl groups located on the ion transport proteins,2) peroxidation of membrane phospholipids, and 3) inhibition ofmembrane-bound regulatory enzymes and modification of the oxidativephosphorylation and ATP levels. Alterations in the ion transportmechanisms lead to changes in a second messenger system, primarilyCa²⁺ homeostasis, which furtheraugment the abnormal electrical activity and distortion of signaltransduction, causing cell dysfunction, which underlies pathologicalconditions.

     

Aberrant cell-to-cell coupling in Ca2+-overloaded guinea pig ventricular muscles

To investigate how intercellular coupling can be changed during Ca²⁺ overloading of ventricular muscle, we studied Ca²⁺ signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential Ca²⁺ images of surface cells were obtained with a confocal microscope. In intact muscles, all cells showed simultaneous Ca²⁺ transients in response to field stimulation over a field of view of 0.3 x 0.3 mm². In severely Ca²⁺-overloaded muscles, obtained by high-frequency stimulation in nonflowing Krebs solution, cells became less responsive to stimulation. Furthermore, nonsimultaneous but serial onsets of Ca²⁺ transients were often detected, suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in Ca²⁺-overloaded muscles, the distributions of phosphorylated and nonphosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the nonoverloaded muscles, whereas nonphosphorylated Cx43 was significantly elevated in severely Ca²⁺-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few square millimeters, can be a cause of abnormal conduction and a microreentry in Ca²⁺-overloaded heart. Inactivation of Na⁺ channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay. Ca²⁺ transient; connexin43; propagation delay; gap junction; arrhythmia     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels     

Aluminium Effects on Pollen Germination and Tube Growth of Chamelaucium uncinatum. A Comparison with Other Ca2+Antagonists

An interaction between aluminium (Al) and calcium (Ca) may bea cause of Al toxicity in plants. The pollen tube is a suitablesystem to test the interaction between Al and Ca since Ca ionsplay a pivotal role in pollen germination and tube growth. Weinvestigated how Al and other known blockers of Ca²⁺-permeablechannels (trivalent cations, ruthenium red, verapamil and nifedipine)influence pollen of an Australian native species Geraldton waxflower(Chamelaucium uncinatum). Pollen germination was inhibited bymicromolar concentrations of trivalent cations (La³⁺>Al³⁺>Gd³⁺)and ruthenium red, but it was relatively insensitive to a micromolarconcentration of verapamil. Exposure of the growing pollen tubesto micromolar concentrations of Al³⁺and La³⁺, and a millimolarconcentration of Ca²⁺chelator ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA) led to rapid tip bursting.In contrast, exposure to Gd³⁺, nifedipine, ruthenium red, verapamiland the organic trivalent cation tris (ethylenediamine)cobalt(TEC³⁺) caused only inhibition of pollen tube growth. The Al³⁺-relatedpollen tube bursting was reduced significantly by increasingeither solution pH from 4.5 to 6 or activity of Ca²⁺from 0.25to 5 m M. In contrast, La³⁺-related pollen tube bursting wasinsensitive to changes in Ca²⁺activity. The results are discussedin terms of Al interactions with cell wall Ca²⁺and the plasmamembrane Ca²⁺-permeable channels. Copyright 1999 Annals of BotanyCompany Aluminium toxicity, Ca²⁺-channel blockers, cell wall, Chamelaucium uncinatum, pollen germination, pollen tube growth.     

Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry