ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.     

Cell death in the anuran tadpole tail: thyroid hormone induces keratinization and tail-specific growth inhibition of epidermal cells

The mechanism of thyroid hormone-induced and glucocorticoid-modulated death of tail epidermal cells from tadpoles of bullfrog, Rana catesbeiana, was investigated by comparing tail epidermal cells with dorsal body epidermal cells. From morphological and biochemical criteria, there were two types of epidermal cells: basal cells and skein cells. The abundance of these cells was different between the tail and the body skin. Fifty percent of body cells and more than 95% of tail cells were skein cells. Effects of 3,3',5-triiodo-L-thyronine (T3, 10(-8) M) and cortisol (5 X 10(-7) M) were investigated with cultured epidermal cells. T3 differently regulated the keratinization of the tail and body cells. The keratinization of the tail epidermal cells was not observed without T3. T3 induced the keratinization dramatically. On the other hand, body epidermal cells were constantly undergoing keratinization without the hormone: T3 merely accelerated the rate of keratinization. Cortisol generally did not show any significant effect on keratinization. T3 showed opposite effects on DNA synthesis of the tail and body cells: suppression of tail cells and stimulation of body cells. Cortisol weakened the inhibitory effect of T3 on DNA synthesis in tail cells. Immunofluorescent micrographs with anti-BrdU showed that T3 decreased the number of cells in the S phase of the cell cycle in the case of tail cells but not of body cells. Thus, thyroid hormone plays dual roles for the tadpole epidermal cells: one is an induction and a promotion of keratinization in tail and body cells, respectively, and the other is an opposite regulation for the proliferation of both epidermal cells. These roles seem to have crucial connections to a tail-specific cell death induced by thyroid hormone.     

On the mechanism of cesium-induced voltage and current tails in single ventricular myocytes

The mechanisms by which different concentrations of cesium modify membrane potentials and currents were investigated in guinea pig single ventricular myocytes. In a dose-dependent manner, cesium reversibly decreases the resting potential and action potential amplitude and duration, and induces a diastolic decaying voltage tail (V_ex), which increases at more negative and reverses at less negative potentials. In voltage-clamped myocytes, Cs⁺ increases the holding current, increases the outward current at plateau levels while decreasing it at potentials closer to resting potential, induces an inward tail current (I_ex) on return to resting potential and causes a negative shift of the threshold for the inward current. During depolarizing ramps, Cs⁺ decreases the outward current negative to inward rectification range, whereas it increases the current past that range. During repolarizing ramps, Cs⁺ shifts the threshold for removal of inward rectification negative slope to less negative values. Cs⁺-induced voltage and current tails are increased by repetitive activity, caffeine (5 mM) and high [Ca²⁺]_o (8.1 mM), and are reduced by low Ca²⁺ (0.45 mM), Cd²⁺ (0.2 mM) and Ni²⁺ (2 mM). Ni²⁺ also abolishes the tail current that follows steps more positive than E_Ca. We conclude that Cs⁺ (1) decreases the resting potential by decreasing the outward current at more negative potentials, (2) shortens the action potential by increasing the outward current at potentials positive to the negative slope of inward rectification, and (3) induces diastolic tails through a Ca²⁺-dependent mechanism, which apparently is an enhanced electrogenic Na-Ca exchange.     

Calcium-mediated modulation of microtubule assembly in human breast epithelial cells

Summary Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca²⁺ medium (1.05 mM). To clarify the mechanism by which Ca²⁺ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca²⁺ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca²⁺ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca²⁺ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca²⁺-grown cells did not display a network of tubulin fibers, whereas high Ca²⁺-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca²⁺ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatement of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca²⁺ medium had no observable effect on cell morphology. These results indicate that high levels of Ca²⁺ either favor tubulin polymerization or stabilize the polymerized state. This research was supported by NCI grant CA-38921 from the National Cancer Institute, Bethesda, MD, and by an institutional grant from the United Foundation of Greater Detroit.     

The growth of WI-38 cells in a serum-free,growth factor-free,medium with elevated calcium concentrations

Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated Ca²⁺ concentrations was investigated. At 5 mM CaCl₂, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl₂ or epidermal growth factor (1 mM CaCl₂) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor and CaCl₂ resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to elevated Ca²⁺ concentrations. In fact, elevated Ca²⁺ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their response to the combined treatment of insulin, transferrin and dexamethasone. This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378. Editor's Statement This paper rigorously dissects the interplay among external Ca²⁺ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan     

Calcium Homeostasis in Digitonin-Permeabilized Bovine Chromaffin Cells

The regulation of cytosolic calcium was studied in digitonin-permeabilized chromaffin cells. Accumulation of ⁴⁵Ca²⁺ by permeabilized cells was measured at various Ca²⁺ concentrations in the incubation solutions. In the absence of ATP, there was a small (10–15% of total uptake) but significant increase in accumulation of Ca²⁺ into both the vesicular and nonvesicular pools. In the presence of ATP, the permeabilized cells accumulated Ca²⁺ into carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-sensitive and -insensitive pools. The CCCP-sensitive pool—mainly mitochondria—was active when the calcium concentration was > 1 μM and was not saturated at 25 μM. The Ca²⁺ sequestered by the CCCP-insensitive pool could be inhibited by vanadate and released by inositol trisphosphate, a combination suggesting that this pool was the endoplasmic reticulum. The CCCP-insensitive pool had a high affinity for calcium, with an EC₅₀ of ～1 μM. When the Ca²⁺ concentration was adjusted to the level in the cytoplasm of resting cells (0.1 μM), the presumed endoplasmic reticulum pool was responsible for ～90% of the ATP-stimulated calcium uptake. At a calcium level similar to the acetylcholine-stimulated level in intact cells (5–10 μM), most of the Ca²⁺ (>95%) went into the CCCP-sensitive pool.     

Darier disease : A disease model of impaired calcium homeostasis in the skin

The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca²⁺ from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca²⁺ transport by SERCA2 resulting in decreased ER Ca²⁺ concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca²⁺ signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Degradation of chlorinated and non-chlorinated aromatic solvents in soil suspensions by pure bacterial cultures

Summary Experiments were performed to test whether or not high concentrations of CaCl₂ (100 mM) are able to arrest and stabilize internal structures and associated functions in Euglena gracilis Z cells stored in darkness at 4° C. Storage of photoheterotrophically grown green cells in high Ca²⁺ media (2–100 mM) retards pheophytinization of the chlorophylls, preserves photosynthetic activities and stabilizes the structural organization of the associated light-harvesting complexes of the photosystem II units. Alterations of photosynthesis and respiration by chlorpromazine or by temperature are strongly reduced in cells stored under such conditions. More precisely, a chlorpromazine inhibition site is evidenced in the mitochondrial electron pathway and its location in the chloroplastic electron pathway is clarified. Adaptation of Euglena cells from 2 mM to 100 mM Ca²⁺ medium is accompanied by an increase both in the externally bound and total internal calcium concentration. A mechanism involving a Ca²⁺ deposit on internal membranes is proposed. Such interpretation is extended to the storage of cells immobilized in Ca²⁺-alginate gel.Nomenclature (Ca²⁺)ex external calcium concentration - Chl chlorophylls - (Cl^–)ex external chloride concentration - CPZ chlorpromazine or 2-chloro-10-(3-dimethylaminopropyl)-phenothiazine - DCMU diuron or (3,4-dichorophenyl)-1,1-dimethylurea - EGTA ethylene glycol-bis(-aminoethylether) N,N,N ,N-tetraacetic acid - Fc initial level of chlorophyll fluorescence with DCMU - Fmax maximal level of chlorophyll fluorescence with DCMU - Fo level of chlorophyll fluorescence after transients - Ft level of chlorophyll fluorescence with DCMU - Pheo pheophytins - PS I and PS II photosystems I and II - SMi storage medium Offprint requests to: C. Tamponnet     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Body-specific proliferation of adult precursor cells in Xenopus larval epidermis

Cell proliferation was examined in the back and tail epidermis of larval Xenopus laevis using bromodeoxyuridine (BrdU). The BrdU labeling index of the back epidermis increased temporally at stage 59, followed by a rapid decrease to the same level as at stage 51. The temporal increase in cell proliferation of the back epidermis produced a new epidermal layer composed of basal cells. In vitro analysis showed that tri-iodothyronine (T₃) promotes cell proliferation of basal cells but suppresses that of skein cells. Immunohistochemical studies showed that the newly formed basal cell layer functions as adult precursor cells which produce the adult epidermal cells. In contrast to the back epidermis, the labeling index of the tail epidermis decreased from stage 57. However, when the tail skin was transplanted to the back area, cell proliferation in the tail epidermis increased to the same level as that of the normal back epidermis. Cell proliferation of the back epidermis was not suppressed by transplanting the skin to the tail area. These results suggest that some promoting factors are produced in the body region and regulate the number of adult precursor cells, which determine the developmental fate of the larval skin.     

Exocytosis Coupled to Mobilization of Intracellular Calcium by Muscarine and Caffeine in Rat Chromaffin Cells

Abstract: We used cultured rat chromaffin cells to test the hypothesis that Ca²⁺ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca²⁺ sources: (a) Ca²⁺ surrounding single cells was transiently displaced by applying agonist with or without Ca²⁺ from an ejection pipette. (b) Intracellular stores of Ca²⁺ were depleted by soaking cells in Ca²⁺-free plus 1 mM EGTA solution before transient exposure to agonist plus Ca²⁺. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 µM) caused an immediate increase in [Ca²⁺]_i and secretion in cells with or without internal Ca²⁺ stores, but only when applied with Ca²⁺ in the ejection pipette. Caffeine (10 mM) and muscarine (30 µM) evoked exocytosis whether or not Ca²⁺ was included in the pipette, but neither produced responses in cells depleted of internal Ca²⁺ stores. Pretreatment with ryanodine (0.1 µM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca²⁺]_i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 ± 0.38 s) or muscarine (2.2 ± 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 ± 0.8 s) was significantly shorter than that evoked by muscarine (33.1 ± 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An ～20-s refractory period was found between repeated caffeine-evoked exocytotic bursts even though [Ca²⁺]_i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca²⁺ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca²⁺. These results and conclusions are consistent with our original observations in the perfused adrenal gland.     

Development of depolarization‐induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K⁺ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K⁺ (1 min) with a transient increase in [Ca²⁺]_i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K⁺, the amplitude of the [Ca²⁺]_i transients averaging 592 nM. At later stages, all cells responded to 25 mM K⁺ with [Ca²⁺]_i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca²⁺]_i transients. The amplitudes of these [Ca²⁺]_i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca²⁺]_i transients were greatly reduced in Ca²⁺‐free, EGTA‐buffered saline, and in the presence of the Ca²⁺ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca²⁺ influx through voltage‐activated Ca²⁺ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca²⁺ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Adult precursor cells in the tail epidermis of Xenopus tadpoles

Polyclonal antibodies were raised against Xenopus larva-specific 58 kDa keratin (PAK58) and adult-specific 63 kDa keratin (PAK63), in order to examine the origin of 63 kDa-keratin-producing cells in the tail skin. By immunofluorescent staining of the tail skin, the 58 kDa keratin was recognized in almost all of the larval epidermal cells, although a small number of PAK58-negative cells were detected at stage 64. In contrast, 63 kDa keratin was immunohistochemically recognized at stage 58, but the signal was very weak. The number of epidermal layers in the tail epidermis increased during a period from stage 58 to stage 64. At stage 64, a small number of PAK63-positive cells was clearly identified in the multilayered tail epidermis. Comparative analysis of successive sections showed that PAK63-positive cells are derived from a cell group differing from PAK58-positive cells. Immunohistochemical studies using cultured epidermal cells demonstrated that 58 kDa keratin is localized in the cytoskeletal bundles of skein cells, whereas 63 kDa keratin is produced not by skein cells but by basal cells and their descendants. These results suggest that basal cells are the adult precursor cells within the larval epidermis even in the tail area.     

Calcium mobilization and muscle contraction induced by acetylcholine in swine trachealis

The roles of Ca²⁺ mobilization in development of tension induced by acetylcholine (ACh, 0.1–100 µM) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca²⁺]_i) that declined to a steady-state level. The peak increase in [Ca²⁺]_i correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca²⁺]_i did not. Removal of extracellular Ca²⁺ had little effect on peak [Ca²⁺]_i but greatly reduced steady-state increases in [Ca²⁺]_i and tension. Verapamil inhibited steady-state [Ca²⁺]_i only at [ACh]<1 µM. After depletion of internal Ca²⁺ stores by 10 min exposure to ACh in Ca²⁺-free solution and then washout of ACh for 5 min in Ca²⁺-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca²⁺ increased [Ca²⁺]_i to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1–100 µM) also reduced the overshoot of [Ca²⁺]_i at 10 and 100 µM ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca²⁺]_i is necessary for tension maintenance and is dependent on Ca²⁺ influx through voltage-gated calcium channels at 0.1 µM ACh and through a verapamil-insensitive pathway at 10 and 100 µM. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists.     

Calcium ionophore-induced egg activation and apoptosis are associated with the generation of intracellular hydrogen peroxide

The present study was designed to investigate whether calcium ionophore-induced activation and apoptosis are associated with the generation of hydrogen peroxide (H₂O₂) in rat eggs cultured in vitro. Culture of metaphase-II (M-II) arrested eggs in Ca²⁺/Mg²⁺-deficient medium did not induce egg activation, while a second polar body was observed in 20% of eggs when cultured in Ca²⁺/Mg²⁺-supplemented medium. In Ca²⁺/Mg²⁺-deficient medium, lower concentrations of calcium ionophore (0.2,0.4 and 0.8 µm) not only induced egg activation in a dose-dependent manner but also generation of intracellular H₂O₂ (84.40±0.50 ng/egg) when compared to control eggs (80.46±1.34 ng/egg). The higher concentration of calcium ionophore (1.6 µm) induced apoptosis and pronounced generation of intracellular H₂O₂ (92.43±0.93 ng/egg) in treated eggs. Conversely, cell-permeant antioxidant such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) reduced intracellular H₂O₂ level (81.20±1.42 ng/egg) and protected against calcium ionophore-induced morphological changes characteristics of egg activation and apoptosis. These results clearly suggest that calcium ionophore-induced activation and apoptosis are associated with the generation of intracellular H₂O₂ in rat eggs.     

Tight junction regulates epidermal calcium ion gradient and differentiation

It is well known that calcium ions (Ca²⁺) induce keratinocyte differentiation. Ca²⁺ distributes to form a vertical gradient that peaks at the stratum granulosum. It is thought that the stratum corneum (SC) forms the Ca²⁺ gradient since it is considered the only permeability barrier in the skin. However, the epidermal tight junction (TJ) in the granulosum has recently been suggested to restrict molecular movement to assist the SC as a secondary barrier. The objective of this study was to clarify the contribution of the TJ to Ca²⁺ gradient and epidermal differentiation in reconstructed human epidermis. When the epidermal TJ barrier was disrupted by sodium caprate treatment, Ca²⁺ flux increased and the gradient changed in ion-capture cytochemistry images. Alterations of ultrastructures and proliferation/differentiation markers revealed that both hyperproliferation and precocious differentiation occurred regionally in the epidermis. These results suggest that the TJ plays a crucial role in maintaining epidermal homeostasis by controlling the Ca²⁺ gradient.     

High Calcium Permeability of Serotonin 5-HT3 Receptors on Presynaptic Nerve Terminals from Rat Striatum

Abstract: The serotonin 5-HT₃ receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT₃ receptors induce Ca²⁺ influx. Whereas postsynaptic 5-HT₃ receptors induce depolarization, being permeant to Na⁺ and K⁺, the basis of presynaptic 5-HT₃ receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT₃ receptor agonist, induced increases in internal free Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca²⁺]_i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT₃ receptor antagonist, but were insensitive to the removal of external free Na⁺ (substituted with N-methyl-d -glucamine), to prior depolarization induced on addition of 20 mM K⁺, or to voltage-gated Ca²⁺ channel blockade by 10 µM Co²⁺/Cd²⁺ or by 1 µMω-conotoxin MVIIC/1 µMω-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca²⁺ influx induced by 5-HT₃ receptor activation in NG108-15 cells by 1 µM mCPBG was substantially reduced by 10 µM Co²⁺/Cd²⁺ and was completely blocked by 1 µM nitrendipine, an L-type Ca²⁺ channel blocker. We conclude that in contrast to the perikaryal 5-HT₃ receptors, presynaptic 5-HT₃ receptors appear to be uniquely calcium-permeant.     

Oleic Acid Blocks Epidermal Growth Factor-Activated Early Intracellular Signals without Altering the Ensuing Mitogenic Response

In EGFR-T17 cells, which express high levels of the epidermal growth factor (EGF) receptor, addition of a saturating dose of EGF (10 nM) leads to an increase in Ins(1,4,5)P₃/diacylglycerol and also to cytosolic calcium [Ca²⁺]_i due to both intracellular redistribution and influx from extracellular medium. Pretreatment of cells with cis -unsaturated nonesterified fatty acids such as oleic acid (1 to 100 μM) inhibited EGF-stimulated Ins(1,4,5)P₃ generation and Ca²⁺ release from intracellular stores. Furthermore, such a treatment completely suppress Ca²⁺ influx in a dose-dependent manner. At doses capable of suppressing such early signals, oleic acid did not alter the process of EGF-mediated internalization of the EGF/EGF-receptor complex, suggesting that [Ca²⁺]_i rise did not mediate receptor internalization. EGF-induced cell proliferation assessed by either thymidine incorporation into DNA, direct cell counting, and microscopic observation was not altered by oleic acid, at doses able to block EGF-mediated early signals. In conclusion, suppression of Ins(1,4,5)P₃ generation and [Ca²⁺]_i rises by oleic acid did not alter EGF-receptor internalization nor EGF-induced cell mitosis. Such results suggest that [Ca²⁺]_i rise is not instrumental for EGF-stimulated cell proliferation.