Small Synthetic Peptides Homologous to Segments of the First External Loop of Occludin Impair Tight Junction Resealing

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca²⁺ removal and was characterized by a marked drop of TER. The reintroduction of Ca²⁺ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca²⁺ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions. Received: 4 December 1998/Revised: 22 January 1999     

Protein Kinase Inhibitors and the Dynamics of Tight Junction Opening and Closing in A6 Cell Monolayers

This study focuses, in A6 cell monolayers, on the role of protein kinases in the dynamics of tight junction (TJ) opening and closing. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺-switch assay (FCSA), which consisted of opening the TJs by removing basolateral Ca⁺⁺ (Ca⁺⁺ _bl), and closing them by returning Ca⁺⁺ _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the evaluation of the effects of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in response to the FCSA followed single-exponential time courses. A rise of apical Ca⁺⁺ (Ca⁺⁺ _ap) causes a reduction of TJ opening rate in an FCSA or even a partial recuperation of G, an effect that is interpreted as mediated by Ca⁺⁺ _ap entering the open TJs. Protein kinase C (PKC) inhibition by H7 at low concentrations caused a reduction of the rate of junction opening in response to Ca⁺⁺ _bl removal, without affecting junction closing, indicating that PKC in this preparation is a key element in the control of TJ opening dynamics. H7 at 100 μm completely inhibits TJ opening in response to Ca⁺⁺ _bl withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase, a process that can be halted again by H7 reintroduction into the bathing solution. Differently from the condition in which Ca⁺⁺ is absent from the apical solution, in which H7 halts the process of G increase in response to a FCSA, when Ca⁺⁺ is present in the apical solution, addition of H7 during G increase in an FCSA not only induces a halt of the G increase but causes a marked recuperation of the TJ seal, indicated by a drop of G, suggesting a cooperative effect of Ca⁺⁺ and H7 on the TJ sealing process. Staurosporine, another PKC inhibitor, differently from H7, slowed both G increase and G decrease in an FCSA. Even at high concentrations (400 nm) staurosporine did not completely block the effect of Ca⁺⁺ withdrawal. These discrepancies between H7 and staurosporine might result from distinct PKC isoforms participating in different steps of TJ dynamics, which might be differently affected by these inhibitors. Immunolocalizations of TJ proteins, carried out in conditions similar to the electrophysiological experiments, show a very nice correlation between ZO-1 and claudin-1 localizations and G alterations induced by Ca⁺⁺ removal from the basolateral solution, both in the absence and presence of H7. Received: 18 April 2001/Revised: 16 July 2001     

Tight Junction Dynamics: Oscillations and the Role of Protein Kinase C

The present study aimed to characterize the role of protein kinase C (PKC) on the dynamics of tight junction (TJ) opening and closing in the frog urinary bladder. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺ switch assay (FCSA), which consisted in opening the TJs by removing basolateral Ca⁺⁺ ([Ca⁺⁺] _bl ), and closing them by returning [Ca⁺⁺] _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the appraisal of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in an FCSA were shown to follow single exponential time courses. PKC inhibition by H7 (100 μm) caused a reduction of the rate of junction opening in response to removing [Ca⁺⁺] _bl , without affecting junction closing, indicating that PKC is a key element in the control of TJ opening dynamics in this preparation. H7 at 250 μm almost completely inhibits TJ opening in response to basolateral Ca⁺⁺ withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase which, however, once started cannot be stopped by H7 reintroduction, Ca⁺⁺ being necessary to allow TJ recovery. A step rise of apical Ca⁺⁺ concentration ([Ca⁺⁺] _ap ) causes a reduction of the rate of TJ opening in a FCSA, an effect that is believed to be mediated by apical Ca⁺⁺ entering the open TJs. The specific condition of having Ca⁺⁺ only in the apical solution and the TJs located midway between the Ca⁺⁺ source (apical solution) and the Ca⁺⁺-binding sites presumably located at the zonula adhaerens, might configure a situation in which a control feedback loop is set up. A rise of [Ca⁺⁺] _ap during the phase of G increase in an FCSA causes a transient recovery of G followed by a subsequent escape phase where G increases again. Oscillations of G also appear in response to a rise of apical Ca⁺⁺. Both escape and oscillations result from the properties of the TJ regulatory feedback loop. In conclusion, the present results indicate that PKC plays a key role in TJ opening in response to extracellular Ca⁺⁺ withdrawal without major effect on the reverse process. In addition, PKC inhibition by H7 not only prevents TJ opening in response to basolateral Ca⁺⁺ removal but induces a prompt blockade of TJ oscillations induced by apical Ca⁺⁺, oscillations which reappear again when H7 is removed. Received: 9 May 2000/Revised: 30 August 2000     

Behavior of tricellulin during destruction and formation of tight junctions under various extracellular calcium conditions

Tricellulin is an important component of tricellular tight junctions (TJs) and is involved in the formation of tricellular contacts. However, little is known about its regulation during the assembly and disassembly of tricellular TJs. By using the well-differentiated pancreatic cancer cell line HPAC, which highly expresses tricellulin at tricellular contacts, we have investigated changes in the localization, expression and phosphorylation of tricellulin and in its TJ functions as a barrier and fence during the destruction and formation of TJs induced by changes in the extracellular calcium concentration. During both extracellular Ca²⁺ depletion caused by EGTA treatment and Ca²⁺ repletion after Ca²⁺ starvation, the expression of tricellulin increased in whole lysates and in Triton-X-100-insoluble fractions without any change in its mRNA. The increases in immunoreactivity revealed by Western blotting were prevented by alkaline phosphatase treatment. Immunoprecipitation assays showed that tricellulin was phosphorylated on threonine residues when it increased after Ca²⁺ depletion and repletion. In the early stage after Ca²⁺ repletion, tricellulin was expressed not only at tricellular contacts but also in the cytoplasm and at bicellular borders. In confocal laser microscopy, tricellulin was observed at the apical-most regions and basolateral membranes of tricellular contacts after Ca²⁺ repletion. Knockdown of tricellulin delayed the recovery of the barrier and fence functions after Ca²⁺ repletion. Thus, the dynamic behavior of tricellulin during the destruction and formation of TJs under various extracellular calcium conditions seems to be closely associated with the barrier and fence functions of TJs.     

Regulation of Ca2+-activated K+ Channel from Rabbit Distal Colon Epithelium by Phosphorylation and Dephosphorylation

The Ca²⁺-activated maxi K⁺ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na⁺ reabsorption from the intestinal lumen. Previous measurements of these basolateral K⁺ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca²⁺ with a K _0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca²⁺. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K⁺ channels lost their high sensitivity to Ca²⁺, yet they could still be activated by high concentrations of Ca²⁺ (10 μm). Furthermore, the high sensitivity to Ca²⁺ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca²⁺ of the maxi K⁺ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca²⁺ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca²⁺ sensitivities for maxi K⁺ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. Received: 28 February 1995/Revised: 22 December 1995     

Regulation of Intracellular Ca2+ by CFTR in Chinese Hamster Ovary Cells

In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl⁻ secretion. As recently proposed, beside its role of Cl⁻ channel, CFTR may regulate the activity of other channels such as a Ca²⁺-activated Cl⁻ channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca²⁺] _i ([Ca²⁺] _i ), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca²⁺] _i in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca²⁺] _i increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca²⁺] _i in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca²⁺] _i response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca²⁺] _i by CFTR in CHO epithelial cell membranes. Received: 5 April 1999/Revised: 28 June 1999     

Tight Junctions and the Experimental Modifications of Lipid Content

Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (J_DEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the J_DEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms. Received: 7 November 1997/Revised: 20 March 1998     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000     

Kinetic Differences in the Phospholamban-Regulated Calcium Pump When Studied in Crude and Purified Cardiac Sarcoplasmic Reticulum Vesicles

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca²⁺ with little or no effect on V _max(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca²⁺ is larger, while the increase in V _max(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca²⁺ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca²⁺ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 μm ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in V _max(Ca) in addition to an increase in the pump's apparent affinity for Ca²⁺ in response to phosphorylation of PLN by protein kinase A. Received: 20 May 1998/Revised: 13 November 1998     

Mechanically Induced Calcium Movements in Astrocytes, Bovine Aortic Endothelial Cells and C6 Glioma Cells

Forces applied to resting primary astrocytes, bovine aortic endothelial cells and C6 glioma cells with collagen-coated magnetite particles produce a fast transient change of intracellular Ca²⁺. It peaks in the micromolar range as measured by Fura-2. This mechanical response adapts within seconds so that repeated stimulation causes smaller responses requiring >10 min for recovery. When cytoplasmic Ca²⁺ is high after treating with ATP, cyclopiazonic acid and thapsigargin, stimulation causes a transient decrease in Ca²⁺. In these three cell types, no influx of ions is required for Ca²⁺ elevation showing the response is not caused by activation of plasmalemmal mechanosensitive channels. Approximately half the cells tested showed similar behavior, while the other half, such as fibroblasts, required extracellular Ca²⁺. The Ca²⁺ response is not temperature sensitive suggesting the possible involvement of intracellular mechanosensitive channels. We tested a number of second messenger reagents and were only able to block the response in BAECs, but not C6 glioma cells, with Xestospongin C, a blocker of IP₃-activated channels. Despite the lack of a causal involvement of plasmalemmal mechanosensitive channels, mechanical stimulation immediately activates a persistent Mn²⁺ influx pathway. This Mn²⁺ pathway may be mechanosensitive channels, Ca²⁺-activated cation channels or depletion-activated Ca²⁺ channels. Received: 7 July 1999/Revised: 12 November 1999     

Calcium Site Specificity : Early Ca2+-related Tight Junction Events

The molecular mechanisms by which Ca²⁺ and metal ions interact with the binding sites that modulate the tight junctions (TJs) have not been fully described. Metal ions were used as probes of these sites in the frog urinary bladder. Basolateral Ca²⁺ withdrawal induces the opening of the TJs, a process that is abruptly terminated when Ca²⁺ is readmitted, and is followed by a complete recovery of the TJ seal. Mg²⁺ and Ba²⁺ were incapable of keeping the TJ sealed or of inducing TJ recovery. In addition, Mg²⁺ causes a reversible concentration-dependent inhibition of the Ca²⁺-induced TJ recovery. The effects of extracellular Ca²⁺ manipulation on the TJs apparently is not mediated by changes of cytosolic Ca²⁺ concentration. The transition elements, Mn²⁺ and Cd²⁺, act as Ca²⁺ agonists. In the absence of Ca²⁺, they prevent TJ opening and almost immediately halt the process of TJ opening caused by Ca²⁺ withdrawal. In addition, Mn²⁺ promotes an almost complete recovery of the TJ seal. Cd²⁺, in spite of stabilizing the TJs in the closed state and halting TJ opening, does not promote TJ recovery, an effect that apparently results from a superimposed toxic effect that is markedly attenuated by the presence of Ca²⁺. The interruption of TJ opening caused by Ca²⁺, Cd²⁺, or Mn²⁺, and the stability they confer to the closed TJs, might result from the interaction of these ions with E-cadherin. Addition of La³⁺ (2 μM) to the basolateral Ca²⁺-containing solution causes an increase of TJ permeability that fully reverses when La³⁺ is removed. This effect of La³⁺, observed in the presence of Ca²⁺ (1 mM), indicates a high La³⁺ affinity for the Ca²⁺-binding sites. This ability of La³⁺ to open TJs in the presence of Ca²⁺ is a relevant aspect that must be considered when using La³⁺ in the evaluation of TJ permeability of epithelial and endothelial membranes, particularly when used during in vivo perfusion or in the absence of fixatives.     

Pulses of Cell Ca<Superscript>2+</Superscript> and the Dynamics of Tight Junction Opening and Closing

A mathematical modeling of tight junction (TJ) dynamics was elaborated in a previous study (Kassab, F., Marques, R.P., Lacaz-Vieira, F. 2002. Modeling tight junction dynamics and oscillations. J. Gen. Physiol. 120:237–247) to better understand the dynamics of TJ opening and closing, as well as oscillations of TJ permeability that are observed in response to changes of extracellular Ca²⁺ levels. In this model, TJs were assumed to be specifically controlled by the Ca²⁺ concentration levels at the extracellular Ca²⁺ binding sites of zonula adhaerens. Despite the fact that the model predicts all aspects of TJ dynamics, we cannot rule out the likelihood that changes of intracellular Ca²⁺ concentration (Ca²⁺ _cell), which might result from changes \ of extracellular Ca²⁺ concentration (Ca²⁺ _extl), contribute to the observed results. In order to address this aspect of TJ regulation, fast Ca²⁺-switch experiments were performed in which changes of Ca²⁺ _cell were induced using the Ca²⁺ ionophore A23187 or thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase. The results indicate that the ionophore or thapsigargin per se do not affect basal tissue electrical conductance (G), showing that the sealing of TJs is not affected by a rise in Ca²⁺ _cell. When TJs were kept in a dynamic state, as partially open structures or in oscillation, conditions in which the junctions are very sensitive to disturbances that affect their regulation, a rise of Ca²⁺ _cell never led to a decline of G, indicating that a rise of Ca²⁺ _cell does not trigger per se TJ closure. On the contrary, always the first response to a rise of Ca²⁺ _cell is an increase of G that, in most cases, is a transient response. Despite these observations we cannot assure that a rise of Ca²⁺ _cell is without effect on the TJs, since an increase of Ca²⁺ _cell not only causes a transient increase of G but, in addition, during oscillations a rise of Ca²⁺ _cell induced by the Ca²⁺ ionophore transiently halted the oscillatory pattern of TJs. The main conclusion of this study is that TJ closure that is observed when basolateral Ca²⁺ concentration (Ca²⁺ _bl) is increased after TJs were opened by Ca²⁺ _bl removal cannot be ascribed to a rise of Ca²⁺ _cell and might be a consequence of Ca²⁺ binding to extracellular Ca²⁺ sites.     

Protein Kinase A Activation Phosphorylates the Rat ClC-2 Cl− Channel but Does Not Change Activity

Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl⁻ channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases. Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl⁻ currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl⁻ channel activity. Received: 20 November 2000/Revised: 28 March 2001     

Cardiac Sarcalumenin: Phosphorylation, Comparison with the Skeletal Muscle Sarcalumenin and Modulation of Ryanodine Receptor

Cardiac sarcoplasmic reticulum (SR) contains an endogenous phosphorylation system that under specific conditions phosphorylates two proteins with apparent molecular masses of 150 and 130 kDa. The conditions for their phosphorylation are as for the skeletal muscle sarcalumenin and the histidine-rich Ca²⁺ binding protein (HCP) with respect to: (i) Ca²⁺ and high concentrations of NaF are required; (ii) phosphorylation is obtained with no added Mg²⁺ and shows a similar time course and ATP concentration dependence; (iii) inhibition by similar concentrations of La³⁺; (iv) phosphorylation is obtained with [γ-³²P]GTP; (v) ryanodine binding is inhibited parallel to the phosphorylation of the two proteins. The endogenous kinase is identified as casein kinase II (CK II) based on its ability to use GTP as effectively as ATP, and its inhibition by La³⁺. The association of CK II with the cardiac SR, even after EGTA extraction at alkaline pH, is demonstrated using antibodies against CK II. The cardiac 130 kDa protein is identified as sarcalumenin based on its partial amino acid sequence and its blue staining with Stains-All. Cardiac sarcalumenin is different from the skeletal muscle protein based on electrophoretic mobilities, immunological analysis, peptide and phosphopeptide maps, as well as amino acid sequencing. Preincubation of SR with NaF and ATP, but not with NaF and AMP-PNP caused strong inhibition of ryanodine binding. This is due to decrease in Ca²⁺- and ryanodine-binding affinities of the ryanodine receptor (RyR) by about 6.6 and 18-fold, respectively. These results suggest that cardiac sarcalumenin is an isoform of the skeletal muscle protein. An endogenous CK II can phosphorylate sarcalumenin, and in parallel to its phosphorylation the properties of the ryanodine receptor are modified. Received: 15 December 1998/Revised: 25 March 1999     

Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca²⁺] _i ) associated with periodic Ca²⁺ oscillations during prolonged ACh applications. The early transient rise in [Ca²⁺] _i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca²⁺ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca²⁺] _i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca²⁺-sensitive K⁺ currents. These K⁺ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca²⁺ from intracellular stores which in turn activate Ca²⁺-dependent (BK-type) K⁺ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000     

The Role of Calcium in the Volume Regulation of Rat Lacrimal Acinar Cells

Earlier studies have suggested a role for Ca²⁺ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca²⁺-dependent ion channels (Kotera & Brown, 1993; Park et al., 1994). The involvement of Ca²⁺ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd³⁺ inhibited RVD, suggesting that Ca²⁺ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca²⁺] _i during exposure to hypotonic solutions. The absence of any changes in [Ca²⁺] _i resulted from the buffering of cytosolic Ca²⁺ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca²⁺ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca²⁺] _i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca²⁺-activated K⁺ channels. On the basis of the presented data, we suggest an involvement of intracellular Ca²⁺ in controlling cell volume in lacrimal acinar cells. Received: 20 February 1998/Revised: 1 May 1998     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999     

Activation of Divalent Cation Influx into S. cerevisiae Cells by Hypotonic Downshift

Subjecting Saccharomyces cerevisiae cells to a hypotonic downshift by transferring cells from YPD medium containing 0.8 m sorbitol to YPD medium without sorbitol induces a transient rapid influx of Ca²⁺ and other divalent cations into the cell. For cells grown in YPD at 37°C, this hypotonic downshift increases Ca²⁺ accumulation 6.7-fold. Hypotonic downshift-induced Ca²⁺ accumulation and steady-state Ca²⁺ accumulation in isotonic YPD medium are differentially affected by dodecylamine and Mg²⁺. The Ca²⁺-influx pathway responsible for hypotonic-induced Ca²⁺ influx may account for about 10–35% of Ca²⁺ accumulation by cells growing in YPD. Ca²⁺ influx is not required for cells to survive a hypotonic downshift. Hypotonic downshift greatly reduces the ability of S. cerevisiae cells to survive a 5-min exposure to 10 mm Cd²⁺ suggesting that mutants resistant to acute Cd²⁺ exposure may help identify genes required for hypotonic downshift-induced divalent cation influx. Received: 14 January 1997/Revised: 20 June 1997     

Caffeine-Induced Ca2+ Transients and Exocytosis in Paramecium Cells. A Correlated Ca2+ Imaging and Quenched-Flow/Freeze-Fracture Analysis

Caffeine causes a [Ca²⁺] _i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca²⁺]^rest _i ∼50 to 80 nm, [Ca²⁺]^act _i rises within ≤3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9–28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca²⁺ spread during the subsequent ≥2 sec. Chelation of Ca²⁺ _o considerably attenuated [Ca²⁺] _i increase. Therefore, caffeine may primarily mobilize cortical Ca²⁺ pools, superimposed by Ca²⁺ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca²⁺-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca²⁺] _i signals were reduced by [Ca²⁺] _o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca²⁺] _i signal was generated by caffeine stimulation (with Ca²⁺ _i and Ca²⁺ _o available). We conclude the following. (i) Caffeine can mobilize Ca²⁺ from cortical stores independent of the presence of Ca²⁺ _o . (ii) To yield adequate signals for normal exocytosis, Ca²⁺ release and Ca²⁺ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca²⁺] _i increase entails caffeine-mediated access of Ca²⁺ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca²⁺] _i -values for membrane fusion and contents discharge. Received: 23 May 1997/Revised: 18 August 1997     

Regulation of Cation-selective Channels in Liver Cells

In liver cells, cation-selective channels are permeable to Ca²⁺ and have been postulated to represent a pathway for receptor-mediated Ca²⁺ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca²⁺], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic [Cl⁻], a physiological response to Ca²⁺-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for regulation of receptor-mediated Na⁺ and Ca²⁺ influx. Received: 8 October 1996