Carbonated apatites obtained by the hydrolysis of monetite: Influence of carbonate content on adhesion and proliferation of MC3T3-E1 osteoblastic cells

The influence of the carbonate content in apatites on the adhesion and the proliferation of MC3T3-E1 osteoblastic cells was investigated. B-type carbonated apatites (DCAps) were prepared by the hydrolysis of monetite (CaHPO₄, DCP) in solutions with a carbonate concentration ranging from 0.001 to 0.075 mol l^?1. Stoichiometric hydroxyapatite (DCAp0) was synthesized in carbonate-free solution. MC3T3-E1 cells were seeded on the compacted DCAps and cell adhesion and proliferation were analysed after 24 h and 7 days, respectively, using a MTS assay and fluorescence microscopy. Cell adhesion tends to increase with increasing carbonate content for carbonate contents between 0 and 6.9 wt.% and levels out to an acceptable value (±50% compared to the control) for carbonate contents between 6.9 and 16.1 wt.%. Only DCAps with a carbonate content equal to or higher than 11% support high cell proliferation comparable to the control. On the latter DCAps, the cells have a spread morphology and form a near-confluent layer. A decrease in charge density and crystallinity at the apatite surface, as well as the formation of more spheroidal crystals with increasing carbonate content, might attribute to changes in composition and three-dimensional structure of the protein adsorption layer and hence to the observed cell behaviour. Consequently, only DCAps with a high carbonate content, mimicking early in vivo mineralization, are possible candidates for bone regeneration.     

Characterization, physicochemical properties and biocompatibility of La-incorporated apatites

In this study, the physicochemical properties and biocompatibilities of La-containing apatites were intensively investigated together with their characterizations in terms of composition, structure, valent state and morphology using X-ray diffraction, Fourier-transform infrared spectra, X-ray photoelectron spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy, respectively. The results indicate that the La³⁺ ion can be incorporated into the crystal lattice of hydroxyapatite resulting in the production of La-incorporated apatites (La_xCa_10−x(PO₄)₆(OH)_2+x−2yO_y□y−x (x  0.5, y < 1 + x/2) or La_xCa_10−x(PO₄)₆O_y□y−x (0.5 < x < 2, y = 1 + x/2)) by high-temperature solid phase synthesis. For La content <20%, the product is composed of the major phase, La_x-OAP, as well as a small amount of tricalcium phosphate, but for a La content of 20%, the product is pure La-incorporated oxyapatite with the formula La₂Ca₈(PO₄)₆O₂ (La₂-OAP, x = 2, y = 2). It is also found that the La content plays important roles in both the physicochemical properties and biocompatibilities of the La-incorporated apatites. In contrast to La-free apatite, La-incorporated apatites possess a series of attractive properties, including higher thermal stability, higher flexural strength, lower dissolution rate, larger alkaline phosphatase activity, preferable osteoblast morphology and comparable cytotoxicity. In particular, the sintered La-incorporated apatite block achieves a maximal flexure strength of 66.69 ± 0.98 MPa at 5% La content (confidence coefficient 0.95), increased 320% in comparison with the La-free apatite. The present study suggests that the La-incorporated apatite possesses application potential in developing a new type of bioactive coating material for metal implants and also as a promising La carrier for further exploring the beneficial functions of La in the human body.     

Accelerated suicidal erythrocyte death in Klotho-deficient mice

Klotho, a membrane protein mainly expressed in parathyroid glands, kidney, and choroid plexus, counteracts aging and increases the life span. Accordingly, life span is significantly shorter in Klotho-deficient mice (klotho ^−/− ) than in their wild-type littermates (klotho ^+/+ ). The pleotropic effects of Klotho include inhibition of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) formation. Vitamin D-deficient diet reverses the shortening of life span in klotho ^−/− mice. In a variety of cells, 1,25(OH)₂D₃ stimulates Ca²⁺ entry. In erythrocytes, increased Ca²⁺ entry stimulates suicidal erythrocyte death, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. The present study explored the putative impact of Klotho on eryptosis. According to Fluo3 fluorescence, cytosolic Ca²⁺ concentration was significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. According to annexin V-binding, phosphatidylserine exposure was significantly enhanced, and according to forward scatter, cell volume significantly decreased in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Energy depletion (13 h glucose depletion) and oxidative stress (35 min 1 mM tert-butyl-hydroxyl-peroxide [tert-BOOH]) increased phosphatidylserine exposure to values again significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Reticulocyte number was significantly increased in klotho ^−/− mice, pointing to enhanced erythrocyte turnover. Vitamin D-deficient diet reversed the enhanced Ca²⁺ entry and annexin V-binding of klotho ^−/− erythrocytes. The present observations reveal a novel function of Klotho, i.e., the at least partially vitamin D-dependent regulation of cytosolic Ca²⁺ activity in and suicidal death of erythrocytes.     

HCO<Subscript>3</Subscript><Superscript>−</Superscript>-dependent transient acidification induced by ionomycin in rat submandibular acinar cells

Ionomycin (IM, 5 μM), which exchanges 1 Ca²⁺ for 1 H⁺, changed intracellular pH (pH_i) with Ca²⁺ entry into rat submandibular acinar cells. IM-induced changes in pH_i consisted of two components: the first is an HCO₃ ⁻-dependent transient pH_i decrease, and the second is an HCO₃ ⁻-independent gradual pH_i increase. IM (1 μM), which activates store-operated Ca²⁺ channels, induced an HCO₃ ⁻-dependent and transient pH_i decrease without any HCO₃ ⁻-independent pH_i increase. Thus, a gradual pH_i increase was induced by the Ca²⁺/H⁺ exchange. The HCO₃ ⁻-dependent and transient pH_i decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na⁺/H⁺ exchange, the Cl⁻/HCO₃ ⁻ exchange, or the Na⁺-HCO₃ ⁻ cotransport induces no transient pH_i decrease. Thapsigargin induced no transient pH_i decrease. Thus, IM, not Ca²⁺ entry, reduced pH_i transiently. IM reacts with Ca²⁺ to produce H⁺ in the presence of \textCO ₂ /\textHCO ₃ ^- :  [ \textH - \textIM ] ^- + \text Ca ²⁺  + \textCO ₂ \rightleftarrows [ \textH-\textCa - \textIM ] ⁺ ·\textHCO ₃ ^- + \textH ⁺ {\text{CO}}_{ 2} /{\text{HCO}}_{ 3}{^{ - }} : \, \left[ {{\text{H}} - {\text{IM}}} \right]^{ - } + {\text{ Ca}}^{ 2+ } \,+ {\text{CO}}_{ 2} \rightleftarrows \left[ {{\text{H}}-{\text{Ca}} - {\text{IM}}} \right]^{ + } \cdot {\text{HCO}}_{ 3}{^{ - } }+ {\text{H}}^{ + } . In this reaction, a monoprotonated IM reacts with Ca²⁺ and CO₂ to produce an electroneutral IM complex and H⁺, and then H⁺ is removed from the cells via CO₂ production. Thus, IM transiently decreased pH_i. In conclusion, in rat submandibular acinar cells IM (5 μM) transiently reduces pH_i because of its chemical characteristics, with HCO₃ ⁻ dependence, and increases pH_i by exchanging Ca²⁺ for H⁺, which is independent of HCO₃ ⁻.     

Endothelial function in aorta segments of apolipoprotein E-deficient mice before development of atherosclerotic lesions

Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE^−/−) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca²⁺]_i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE^−/− and C57BL/6J (WT) mice were evoked by phenylephrine (3 × 10⁻⁹–3 × 10⁻⁵ M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca²⁺]_i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE^−/− mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca²⁺]_i in apoE^−/− strips with 10⁻⁶ M cyclopiazonic acid or 10⁻⁷ M Ca²⁺-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE^−/− and WT aorta segments. Basal [Ca²⁺]_i was significantly lower in apoE^−/− than in WT strips. Relaxation by ACh (3 × 10⁻⁹–10⁻⁵ M) was time- and dose-dependently related to [Ca²⁺]_i, but neither ACh-induced relaxation nor Ca²⁺ mobilization were diminished in apoE^−/− strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE^−/− mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca²⁺]_i. These findings further point to important differences between basal and stimulated eNOS activity.     

The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression

The conventional biomimetic apatite coating process can be accelerated by immersing substrates into concentrated simulated body fluid (5 x SBF) at 37 degrees C to form an initial coating of apatite precursor spheres, and transform the precursors into plate-like apatite structures. Depending on processing parameters, different apatite structures can be created over the same substrate. The purpose of this study is to investigate the effects of the different apatite microenvironment on cell spreading, viability, proliferation, and gene expression. MC3T3-E1 preosteoblasts were cultured on five surfaces: conventional apatite (CA), precursor apatite spheres (PreA), large plate-like apatites (LgA), small plate-like apatites (SmA), and tissue culture grade polystyrene (TCPS). PreA induced significantly higher cell death during the first two weeks. TCPS supported more uniform spreading (1 day) and higher proliferation (2 weeks) than CA, LgA, and SmA. Apatites restricted spreading and promoted the extension of cellular projections along the textured surfaces under confocal microscopy observation. By 3 weeks, LgA induced highest expression of mature osteogenic markers osteocalcin (OCN) and bone sialoprotein (BSP) in both regular and osteogenic culture media based on quantitative real-time RT-PCR. The results of this study suggest differential cell responses to subtle changes in apatite microenvironment.     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

Extracellular zinc stimulates a calcium-activated chloride conductance through mobilisation of intracellular calcium in renal inner medullary collecting duct cells

We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn²⁺ on whole-cell Ca²⁺-activated Cl⁻ currents (I _CLCA) in mouse inner medullary collecting duct cells (mIMCD-3). I _CLCA was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10–400 μM) to the bathing solution resulted in a dose-dependent increase in I _CLCA with little change in Cl⁻ selectivity or biophysical characteristics, whereas gadolinium chloride (30 μM) and lanthanum chloride (100 μM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn²⁺ (400 μM) stimulated an increase in intracellular Ca²⁺ to an elevated plateau. The Zn²⁺-stimulated [Ca²⁺]_i increase was inhibited by thapsigargin (200 nM), the IP₃ receptor antagonist 2-aminoethoxydiphenyl borate (10 μM) and removal of bath Ca²⁺. Pre-exposure to Zn²⁺ (400 μM) markedly attenuated the ATP (100 μM)-stimulated [Ca²⁺]_i increase. These data are consistent with the hypothesis that extracellular Zn²⁺ stimulates an increase in [Ca²⁺]_i by a release of calcium from thapsigargin/IP₃ sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca²⁺-sensing receptor, in IMCD cells is discussed.     

Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

Ryanodine receptor 1 (RyR1) is well-known to be expressed in systemic and pulmonary vascular smooth muscle cells (SMCs); however, its functional roles remain largely unknown. In the present study, we attempted to determine the potential importance of RyR1 in membrane depolarization-, neurotransmitter-, and hypoxia-induced Ca²⁺ release and contraction in pulmonary artery SMCs (PASMCs) using RyR1 homozygous and heterozygous gene deletion (RyR1^−/− and RyR1^+/−) mice. Our results indicate that spontaneous local Ca²⁺ release and caffeine-induced global Ca²⁺ release are significantly reduced in embryonic RyR1^−/− and adult RyR^+/− cells. An increase in [Ca²⁺]_i following membrane depolarization with high K⁺ is markedly attenuated in RyR1^−/− and RyR1^+/− PASMCs in normal Ca²⁺ or Ca²⁺-free extracellular solution. Similarly, muscle contraction evoked by membrane depolarization is reduced in RyR1^+/− pulmonary arteries in the presence or absence of extracellular Ca²⁺. Neurotransmitter receptor agonists and inositol 1,4,5-triphosphate elicit a much smaller increase in [Ca²⁺]_i in both RyR1^−/− and RyR1^+/− cells. We have also found that neurotransmitter-evoked muscle contraction is significantly inhibited in RyR1^+/− pulmonary arteries. Hypoxia-induced increase in [Ca²⁺]_i and contraction are largely blocked in RyR1^−/− and/or RyR1^+/− PASMCs. Collectively, our findings provide genetic evidence for the functional importance of RyR1 in spontaneous local Ca²⁺ release, and membrane depolarization-, neurotransmitter-, as well as hypoxia-induced global Ca²⁺ release and attendant contraction in PASMCs.     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Disruption of the Gardos channel (KCa3.1) in mice causes subtle erythrocyte macrocytosis and progressive splenomegaly

Gardos channel, the erythrocyte Ca²⁺-activated K⁺ channel (K_Ca3.1), is considered a major regulator of red blood cell (RBC) volume by mediating efflux of potassium and thus cell dehydration and shrinkage. However, the functional importance of K_Ca3.1 in RBC in vivo is incompletely understood. Here, we used K_Ca3.1^−/−-mice to investigate the consequences of K_Ca3.1 deficiency for RBC indices, functions, and sequestration. RBCs of K_Ca3.1^−/−-mice of all ages were mildly macrocytic but their biconcave appearance being preserved. RBC number, total hemoglobin, and hematocrit were unchanged in the adult K_Ca3.1^−/−-mice and increased in the premature K_Ca3.1^−/−-mice. Filterability, Ca²⁺-dependent volume decrease and osmotic tolerance of RBCs lacking K_Ca3.1 were noticeably reduced when compared to RBC of wild-type littermates. Deformability to increasing shear stress was unchanged. Strikingly, K_Ca3.1^−/−-mice developed progressive splenomegaly which was considerable (∼200% of controls) in the >6-month-old mice and was paralleled by increased iron deposition in the aged mice presumably as a consequence of enhanced RBC sequestration. Daily injections of the K_Ca3.1-blocker TRAM-34 (120 mg/kg) also produced mild splenomegaly in wild-type mice. We conclude that genetic deficit of erythroid K_Ca3.1 causes mild RBC macrocytosis, presumably leading to reduced filterability, and impairs volume regulation. These RBC defects result in mild but progressive splenomegaly. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ivica Grgic and Brajesh P. Kaistha contributed equally to this study.     

Differential actions of AlF 4 − and vanadate on canine trachealis muscle

Fluoride (F⁻) a known stimulator of G-protein, has been reported to inhibit “P”-type ATPase activity in smooth muscles. On the other hand, vanadate, a strong “P”-type ATPase inhibitor, has been reported to stimulate G-protein in some cells. This study was designed to compare the contractile actions of fluoroaluminate (AlF ₄ ⁻ ) and vanadate and to clarify their mechanisms of actions by measuring changes in the amount of cyclic adenosine monophosphate (cAMP) and inositol phosphates. F⁻ and vanadate induced strong contractions in canine trachealis muscle. The F⁻-induced contraction was potentiated by the addition of aluminum (Al³⁺, 20 μM) and inhibited by deferoxamine (200 μM), a heavy metal chelator. Ca²⁺ removal and 10 μM verapamil inhibited the contraction induced by AlF ₄ ⁻ and vanadate. AlF ₄ ⁻ and vanadate increased ⁴⁵Ca influx in the absence and presence of verapamil. AlF ₄ ⁻ -induced contractions were partially relaxed by isoproterenol (38.2±7.4%) in contrast with those induced by vanadate (72.1±5.3%), which could be explained by a decrease of tissue cAMP content by AlF ₄ ⁻ in forskolin-pretreated tissues. Vanadate increased inositol phosphate accumulation as did AlF ₄ ⁻ , although the magnitude of the increase was smaller than that produced by AlF ₄ ⁻ . The increases of inositol phosphate content by both drugs were not affected after the pretreatment by pertussis toxin. These results indicate that, in canine trachealis muscle, AlF ₄ ⁻ elicits contractions by Ca²⁺ influx through potential-operated and -independent Ca²⁺ channels and intracellular Ca²⁺ release by inositol 1,4,5-triphosphate (InsP ₃), which are mediated by stimulation of pertussis-toxin-insensitive G-proteins, and that the mechanisms of the vanadate-induced contraction is different from that of AlF ₄ ⁻ , although it may be partly explained by the stimulation of G-proteins.     

Vasomotion has chloride-dependency in rat mesenteric small arteries

The possibility that Ca²⁺-activated Cl⁻ conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I _Cl(Ca)) and cGMP-dependent (I _Cl(Ca,cGMP)) chloride conductances are important. The effect of anion substitution and Cl⁻ channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl⁻ _o replacement with aspartate blocked vasomotion while 36mM SCN⁻ _o (substituted for Cl⁻) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca²⁺]_i disappeared with 36mM SCN⁻. DIDS and Zn²⁺ blocked I _Cl(Ca,cGMP) but not I _Cl(Ca). Vasomotion was not sensitive to DIDS and Zn²⁺, and DIDS and Zn²⁺ induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn²⁺ was sensitive to 36mM SCN⁻ _o. The anion substitution data indicate that Cl⁻ is crucial for the V _m and [Ca²⁺]_i oscillations underlying vasomotion. The Cl⁻ channel blocker data are consistent with both CaCCs being important.     

A calcium-dependent chloride current in insulin-secreting βTC-3 cells

 Ca²⁺-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting β cells in response to high plasma glucose. A Maxi K⁺ channel has received the most attention, while a low-conductance Ca²⁺-activated K⁺ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca²⁺-dependent Cl^–current [I _Cl(Ca)] in insulin-secreting pancreatic β cells. The reported I_Cl(Ca) could be activated under physiological Ca²⁺ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca²⁺ influx: their current/voltage (I/V) relations followed that of the ”L-like” Ca²⁺ current (I _Ca) present in these cells; they were blocked completely by the removal of external Ca²⁺ or application of Cd²⁺ at concentrations sufficient for complete block of I _Ca; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of I _Ca showed a weak linear voltage dependence, while at voltages positive to activation of I _Ca it followed the voltage dependence of I _Ca. This Ca²⁺-dependent current reversed at –21.5 mV and when the external Cl^–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation for a Cl^–-selective current. Cl^–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was a Ca²⁺-dependent Cl^–current [I _Cl(Ca)]. From substitution of the external Cl^–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for I _Cl(Ca): I ^–>NO₃ ^–>Br^–>Cl^–>Acetate^–. Received: 10 October 1996 / Received after revision and accepted: 19 December 1996     

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca²⁺ during action potentials. Indeed, Ca²⁺-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K⁺ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca²⁺) Ca²⁺ concentrations. Despite their low open probability under these conditions (V₅₀ of +146.8 mV), BK channels were responsible for more than 25% of the total K⁺ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K⁺ channels. The other main source of fast voltage-dependent K⁺ efflux at such a low Ca²⁺ was a transient K⁺ (I_A-type) current activating with V ₅₀ = −14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca²⁺. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca²⁺ concentration, and suggest that they could do so also in the presence of extracellular Ca²⁺, before Ca²⁺ entering the cell facilitates their activity.     

Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells

Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (K _d = 15.4 ± 4.6 μM; B _max = 1.7 ± 0.5 nmol mg⁻¹ protein); and a low affinity site (K _d = 170 ± 94.5 μM; B _max = 14.2 ± 3.9 nmol mg⁻¹ protein). In myotube membranes only a low affinity binding site for GTP (K _d = 169 ± 39 μM; B _max = 12.3 ± 1.4 nmol mg⁻¹ protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca²⁺]_i. The patterns of Ca²⁺ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca²⁺ responses: (1) a fast increase in [Ca²⁺]_i, followed by a sustained [Ca²⁺]_i elevation, and (2) a slow raising and steady prolonged increase in [Ca²⁺]_i. In myotubes, however only fast Ca²⁺ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca²⁺]_i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca²⁺ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

Simultaneous recording of ATP-sensitive K+ current and intracellular Ca2+ in anoxic rat ventricular myocytes. Effects of glibenclamide

We investigated the temporal relationship between the adenosine triphosphate-sensitive K current (K _ATP current), hypoxic shortening and Ca accumulation in cardiomyocytes exposed to anoxia or metabolic inhibition. Whole-cell, patch-clamp experiments were performed with nonstimulated isolated rat heart ventricular muscle cells loaded with the Ca-sensitive fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid (fura-2) via the patch pipette. After approximately 8 min anoxia, the K _ATP current started to rise and reached a maximum of 21.3 ± 3.7 nA (n = 5, recorded at 0 mV clamp potential) within 1–3 min. At that time hypoxic contracture also occurred. Resting cytoplasmic free calcium (Ca_i) did not change significantly before hypoxic shortening. After hypoxic contracture, the K _ATP current decreased and Ca_i started to rise, reaching about 1 μmol/l. The presence of glibenclamide (10 μmol/l) in the bath reduced the anoxia-induced K _ATP current by more than 50%, but did not significantly influence the time dependence of current, hypoxic shortening and Ca_i, or the magnitude of Ca_i. Metabolic inhibition with 1.5 mmol/l CN resulted in K _ATP current increase and hypoxic shortening, occurring somewhat earlier than under anoxia, but all other parameters were comparable. In non-patch-clamped cells loaded with fura-2 AM ester and field-stimulated with 1 Hz, 1 μmol/l glibenclamide had no significant effect on the magnitude of the Ca_i increase caused by exposure of the cells to 1.5 mmol/l CN⁻. After CN⁻ wash-out in non-patch-clamped cells, Ca_i declined, oscillated and finally returned to control values. It can be concluded that glibenclamide inhibits anoxia-induced K _ATP currents only partially and has no significant effect on anoxia-induced rise in resting Ca_i. Received: 3 November 1995/Received after revision: 9 January 1996/Accepted: 16 January 1996     

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

HCO 3 − -dependent volume regulation in α-cells of the rat endocrine pancreas

Ion transport activity in pancreatic α-cells was assessed by studying cell volume regulation in response to anisotonic solutions. Cell volume was measured by a video imaging method, and cells were superfused with either 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid-buffered or HCO₃⁻-buffered solutions. α-Cells did not exhibit a regulatory volume increase (RVI) in response to cell shrinkage caused by hypertonic solutions. A RVI was observed, however, in cells that had first undergone a regulatory volume decrease (RVD), but only in HCO₃⁻-buffered solutions. RVI was also observed in response to a HCO₃⁻-buffered hypertonic solution in which the glucose concentration was increased from 4 to 20 mM. The post-RVD RVI and the glucose-induced RVI were both inhibited by 10 μM 5-(N-methyl-N-isobutyl) amiloride or 100 μM 2,2′-(1,2-ethenediyl) bis (5-isothio-cyanatobenzenesulfonic acid), but not by 10 μM benzamil nor 10 μM bumetanide. These data suggest that Na⁺–H⁺ exchangers and Cl⁻–HCO₃⁻ exchangers contribute to volume regulation in α-cells.     

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

 Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba²⁺ (holding potential, V _h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca²⁺ channels; T-type Ca²⁺ channels were not seen. By using supramaximal concentrations of selective Ca²⁺ channel blockers, the whole-cell I _Ba could be fractionated into various subcomponents. Thus, I _Ba had a 25% fraction sensitive to 1 μM nifedipine (L-type channels), 21% sensitive to 1 μM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 μM ω-agatoxin IVA (P/Q-type channels). The activation of I _Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 μM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of I _Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca²⁺ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba²⁺. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca²⁺ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca²⁺ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates. Received: 1 May 1998 / Received after revision and accepted: 21 May 1998