Effects of hypoxia on stimulus-release coupling mechanisms in cultured bovine adrenal chromaffin cells

Summary To clarify the effects of hypoxia on stimulus-release coupling, we have examined the effects of hypoxia on nicotine-induced catecholamine (noradrenaline and adrenaline) release from, and ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in, cultured bovine adrenal chromaffin cells. Experiments were carried out in media pre-equilibrated with 21% O₂/79% N₂ (control) or with 0% O₂/100% N₂ (hypoxia). Cells were stimulated with either nicotine (activating nicotinic acetylcholine (ACh) receptors) or a high K⁺ concentration (55 mmol/1 KCI; directly activating voltage-dependent Ca²⁺ channels). Hypoxia reduced both nicotine- and high K⁺-induced catecholamine releases from the cells, but the reduction of the former (to about 30% of the control value) was more pronounced than that of the latter (to about 40% of the control value). Nicotine-induced ²²Na⁺ influx, which is considered to reflect the function of nicotinic ACh receptors, was inhibited by hypoxia. Both nicotine- and high K⁺-induced ⁴⁵Ca²⁺ influx into the cells were reduced by hypoxia, but the reduction of the former was more pronounced than that of the latter. Nicotine- and high K⁺-induced increases in [Ca²⁺]_i were reduced by hypoxia to about 30% and 40% of the control values, respectively. These results suggest that hypoxia reduces cation influxes (Na⁺ and Ca²⁺) through both the ligand-gated cation channels of the nicotinic ACh receptor and the voltage-dependent Ca²⁺ channels. Correspondence to K. Lee at the present address     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

Correlation between catecholamine release and sodium pump inhibition in the perfused adrenal gland of the cat

1 Ca²⁺ reintroduction to retrogradely perfused and ouabain (10^-4 M)-treated cat adrenal glands caused a catecholamine secretory response which was greater the longer the time of exposure to the cardiac glycoside. Such a response was proportional to the external Na⁺ concentration [Na⁺]_o.

2 A qualitatively similar, yet smaller response was observed when glands were perfused with Krebs solution lacking K⁺ ions; thus, K⁺ deprivation mimicked the secretory effects of ouabain. Catecholamine secretion evoked by Ca²⁺ reintroduction in K⁺-free solution (0-K⁺) was also proportional to [Na⁺]_o and greater the longer the time of exposure of the gland to 0-K⁺ solution.

3 The ionophore X537A also mimicked the ouabain effects, since Ca²⁺ reintroduction to glands treated with this agent (25 μM) caused a sharp secretory response. When added together with X537A, ouabain (10^-4 M) did not modify the response to the ionophore.

4 N-ethylmaleimide (NEM), another Na⁺, K⁺-ATPase inhibitor, did not evoke the release of catecholamines; on the contrary, NEM (10^-4 M) inhibited the catecholamine secretory response to high [K⁺]_o, acetylcholine, Ca²⁺ reintroduction and ouabain.

5 Ouabain (10^-4 M) inhibited the uptake of ⁸⁶Rb into adreno-medullary tissue by 60%. Maximal inhibition had already occurred 2 min after adding the drug, indicating a lack of temporal correlation between ATPase inhibition and the ouabain secretory response, which took longer (about 30-40 min) to reach its peak. NEM (10^-4 M) blocked ⁸⁶Rb uptake in a similar manner.

6 The results are further evidence in favour of the presence of a Na⁺-Ca²⁺ exchange system in the chromaffin cell membrane, probably involved in the control of [Ca²⁺]_i and in the modulation of catecholamine secretion. This system is activated by increasing [Na⁺]_i, either directly (ionophore X537A, increased [Na⁺]_o) or indirectly (Na⁺ pump inhibition). However, the simple inhibition of Na⁺ pumping does not always lead to a catecholamine secretory response; such is the case for NEM.

     

Stimulation of Erythrocyte Cell Membrane Scrambling by Nystatin

The antifungal ionophore nystatin dissipates the Na⁺ and K⁺ gradients across the cell membrane, leading to cellular gain of Na⁺ and cellular loss of K⁺. The increase of cellular Na⁺ concentration may result in Ca²⁺ accumulation in exchange for Na⁺. Increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i) and loss of cellular K⁺ foster apoptosis‐like suicidal erythrocyte death or eryptosis, which is characterised by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. The present study explored whether nystatin stimulates eryptosis. Cell volume was estimated from forward scatter (FSC), phosphatidylserine exposure from annexin V binding and [Ca²⁺]_i from Fluo3‐fluorescence in flow cytometry. A 48‐hr exposure to nystatin (15 μg/ml) was followed by a significant increase of [Ca²⁺]_i, a significant increase of annexin V binding and a significant decrease of FSC. The annexin V binding after nystatin treatment was significantly blunted in the nominal absence of extracellular Ca²⁺. Partial replacement of extracellular Na⁺ with extracellular K⁺ blunted the nystatin‐induced erythrocyte shrinkage but increased [Ca²⁺]_i and annexin V binding. Nystatin triggers cell membrane scrambling, an effect at least partially due to entry of extracellular Ca²⁺.     

Effects of P2 Purinoceptor Agonists on Membrane Potential and Intracellular Ca2+ of Human Cardiac Endothelial Cells

Abstract: Vasoactive agonists like adenosine-5′-triphosphate (ATP) increase intracellular Ca²⁺ ([Ca²⁺]_i) in vascular endothelial cells with an initial peak due to inositol 1,4,5-triphosphate-mediated Ca²⁺ release from intracellular stores followed by a sustained plateau that is dependent on the presence of extracellular Ca²⁺, thus leading to an increased synthesis and release of prostacyclin and nitric oxide. We studied the effects of nucleotides on membrane potential and [Ca²⁺]_i in confluent human microvascular cardiac endothelial cells obtained from patients with dilated cardiomyopathy. The whole-cell configuration of the patch-clamp technique and a confocal laser scanning microscope employing fluo-3 as a Ca²⁺ indicator were used. Both uridine-5′-triphosphate (UTP) and 2-methylthioadenosine-5′-triphosphate (2MeSATP) induced depolarizations in human microvascular cardiac endothelial cells and increased [Ca²⁺]_i with a rank order of potency 2MeSATP>ATP=UPP (EC₅₀ values (in μM) were 0.084 2MeSATP, 0.67 ATP and 1.1 UTP). This suggests that both P_2u and P_2y purinoceptors are present on human microvascular cardiac endothelial cells. Maximal [Ca²⁺]_i responses of confluent human microvascular cardiac endothelial cell monolayers to UTP were lower when compared to 2MeSATP. Nucleotide-induced increases in [Ca²⁺]_i consisted of a transient peak, which was also observed in the absence of extracellular Ca²⁺, and a sustained [Ca²⁺]_i plateau. This plateau, which was not observed in all monolayers studied, was not markedly influenced by increasing extracellular [K⁺]. Previous incubation with thapsigargin abolished ATP-induced increases of [Ca²⁺]_i. It is concluded that human microvascular cardiac endothelial cells express both P_2y and P_2u purinoceptors. P₂ purinoceptor agonists release Ca²⁺ from intracellular thapsigargin-sensitive stores and stimulate capacitative Ca²⁺ influx pathways. K⁺ efflux through Ca²⁺-dependent K⁺ (K_ca) channels does not play a major role in the regulation of nucleotide-induced Ca²⁺ influx in human microvascular cardiac endothelial cells, which might be related to an impaired function of the cells.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

N,N-dimethyl-D-erythro-sphingosine increases intracellular Ca2+ concentration via Na+-Ca2+-exchanger in HCT116 human colon cancer cells

N,N-dimethyl-D-erythro-sphingosine (DMS), an N-methyl derivative of sphingosine, is an inhibitor of protein kinase C (PKC) and sphingosine kinase (SK). In previous reports, DMS-induced intracellular Ca²⁺ increase concentration ([Ca²⁺]_i) was studied in T lymphocytes, monocytes, astrocytes and neuronal cells. In the present study, we studied DMS-induced increase of [Ca²⁺]_i in HCT116 human colon cancer cells. We found that the DMS-induced increase of [Ca²⁺]_i in colon cancer cells is composed of Ca²⁺ release from intracellular Ca²⁺ stores and subsequent Ca²⁺ influx. The Ca²⁺ release is not related to modulation of inositol 1,4,5-trisphosphate (IP₃) receptor or ryanodine receptor. On the other hand, the Ca²⁺ influx is mediated largely through Ca²⁺ channels sensitive to verapamil, nifedipine, Ga³⁺, and La³⁺. Furthermore, we found that the response is inhibited by bepridil and Ni²⁺, specific inhibitors of Na⁺-Ca²⁺-exchanger, suggesting involvement of Na⁺-Ca²⁺ exchanger in the DMS-induced [Ca²⁺]_i increase in colon cancer cells. This inhibition was also observed in U937 monocytes, but not in 1321N1 astrocytes.     

Involvement of different calcium channels in K+- and veratridine-induced increases of cytosolic calcium concentration in rat cerebral cortical synaptosomes

Intracellular calcium ion concentrations ([Ca²⁺]_i) in rat cerebral cortical synaptosomes were measured, using the calcium chelating fluorescence dye fura-2. The synaptosomes were depolarized by elevation of the extracellular K⁺ concentration or by addition of veratridine, which opens voltage-dependent Na⁺-channels and prevents their inactivation. Both enhancement of the concentration of extracellular K⁺ (up to 60 mM) and veratridine (1–100 μM) increased the [Ca²⁺]_i in a concentration-dependent manner. In the absence of extracellular Ca²⁺, the K⁺- and veratridine-induced increases in [Ca²⁺]_i were abolished, indicating that the increase in [Ca²⁺]_i was due to an influx of extracellular Ca²⁺. Tetrodotoxin (TTX), a blocker of the voltage-dependent Na⁺ channel, inhibited the veratridine-induced (10 μM) Ca²⁺ influx by more than 80%, while the K⁺-evoked (30 mM) increase of [Ca²⁺]_i was TTX-resistant. Both the K⁺- and the veratridine-induced Ca²⁺ influx were not reduced by nifedipine (1 μM), a blocker of L-type Ca²⁺ channels. Blockade of the voltage dependent N-type Ca²⁺ channels with ω-conotoxin GVIA (ω-CTx GVIA; 0.1 μM) and of the voltage-dependent P/Q-type channels with ω-agatoxin IVA (ω-AgaTx IVA; 0.2 μM) inhibited the K⁺-induced increase in [Ca²⁺]_i by about 30 and 55%, respectively; these effects were additive. ω-Conotoxin MVIIC (ω-CTx MVIIC) at a concentration of 0.2 μM, which may be assumed to block predominantly the Q-type Ca²⁺ channel, inhibited the K⁺-induced increase in [Ca²⁺]_i by 50%. The veratridine-induced increase in [Ca²⁺]_i was reduced by about 25% by ω-CTx GVIA (0.1 μM), but was resistant to ω-AgaTx IVA (0.2 μM) and ω-CTx MVIIC (0.2 μM). Mibefradil (former designation Ro 40-5967), a Ca²⁺ antagonist which blocks all types of voltage-dependent Ca²⁺ channels including the T and R channels, led to a concentration-dependent inhibition of the K⁺- and veratridine-induced increase in [Ca²⁺]_i (abolition at 10 μM mibefradil). Ifenprodil, another non-specific blocker of voltage-dependent Ca²⁺ channels, also inhibited the K⁺- and veratridine-induced increase in [Ca²⁺]_i in concentration-dependent manner and abolished it at 320 μM ifenprodil. In contrast, KB-R 7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate; 1 and 3 μM), a highly potent and selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX1), failed to inhibit the K⁺- and veratridine-induced increase in [Ca²⁺]_i. It is concluded that the K⁺-induced increase in free cytosolic Ca²⁺ results from Ca²⁺ influx through voltage-dependent N- and, above all, Q-type Ca²⁺ channels. N-type Ca²⁺ channels also play a minor role in the veratridine-induced increase in [Ca²⁺]_i, but P/Q-type channels do not appear to be involved at all. The inhibition of the veratridine-induced, ω-CTx GVIA- and ω-AgaTx IVA-resistant increase in [Ca²⁺]_i by mibefradil and the failure of KB-R 7943 to inhibit this response are compatible with the suggestion that in rat cerebral cortical synaptosomes, Ca²⁺ influx via the R-type Ca²⁺ channel and/or another so far uncharacterized Ca²⁺ channel may substantially contribute to the veratridine-induced increase in [Ca²⁺]_i. Received: 7 March 1997 / Accepted: 9 September 1997     

Calcium signalling by G protein-coupled sphingolipid receptors in bovine aortic endothelial cells

Besides its role as a putative second messenger releasing Ca²⁺ from intracellular stores, sphingosine-1-phosphate (SPP) has recently been identified as an extracellularly acting ligand activating a high affinity G protein-coupled membrane receptor in various cell types. Since SPP can be released from activated platelets, we examined in the present study whether endothelial cells express receptors for SPP and related sphingolipids. In bovine aortic endothelial cells loaded with fura-2, addition of SPP caused a rapid and transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), amounting to maximally about 230 nM. Removal of extracellular Ca²⁺ revealed that SPP-induced [Ca²⁺]_i elevations were due to both release of Ca²⁺ from intracellular stores and influx of extracellular Ca²⁺. Pretreatment of the cells with pertussis toxin inhibited the SPP-induced increase in [Ca²⁺]_i by 83%, in line with the previously reported involvement of G proteins of the G_i/o family in SPP signalling in other cell types. In contrast to other [Ca²⁺]_i-elevating agonists, e.g., ATP and bradykinin, SPP did not activate phospholipase C in bovine aortic endothelial cells, suggesting the involvement of a novel, unidentified signalling pathway in SPP-induced release of intracellular Ca²⁺. Furthermore, SPP also did not cause activation of either phospholipase D or A₂. Out of various related sphingolipids studied, only sphingosylphosphorylcholine (SPPC) induced a similar maximal increase in [Ca²⁺ _i as SPP, and its effect was also fully pertussis toxin-sensitive. However, the potencies of the two sphingolipids to increase [Ca²⁺]_i differed by more than two orders of magnitude, with the EC₅₀ values being 0.8 nM and 260 nM for SPP and SPPC, respectively. These results identify SPP and SPPC as novel and potent endothelial agonists, inducing calcium signalling by activation of a G_i/o protein-coupled receptor(s). Given the recently reported release of SPP from thrombin-activated platelets, SPP may represent a novel mediator of platelet-endothelial cell interactions.     

Effects of the Chinese herb component phellopterin on the increase in cytosolic free calcium in PC12 cells

The present study investigated the effects of the Chinese Herb component, phellopterin on high K⁺ and glutamate‐induced extracellular calcium influx and caffeine or cyclopiazonic acid (CPA)‐induced calcium release from internal stores in attached PC12 cells. Attached cells were loaded with the calcium fluorescent indicator Fluo‐3/AM with the final concentration of 5 µM for 50 min at 37°C and cytosolic free Ca²⁺ measured as fluorescent intensity (FI) (excitation: 488 nm; emission: 535 nm). When PC12 cells were exposed to extracellular Ca²⁺([Ca²⁺]₀) 2.0 mM, the FI for resting [Ca²⁺]_i was 1,188±163, high K⁺ (75 mM) and glutamate (10 mM) induced an increase in [Ca²⁺]_i with peak values of 4,270±982 and 3,096±402, respectively. Phellopterin (0.1–100 µM) had no apparent effect on resting [Ca²⁺]_i, but inhibited high K⁺ and glutamate induced the increase in [Ca²⁺]_i in a dose‐dependent manner. When PC12 cells were exposed to Ca²⁺‐free solution, the FI for resting [Ca²⁺]_i was 804±77. Caffeine (40 mM) and CPA (30 µM) stimulated Ca²⁺ release from caffeine‐ryanodine and inositol 1,4,5‐tris‐phosphate (InsP₃₎‐sensitive internal calcium stores, inducing an increase in [Ca²⁺]_i to 2,938±362 and 1,816±291, respectively. Phellopterin (0.1–100 µmol/L) inhibited caffeine and CPA stimulated intracellular calcium release in a dose‐dependent manner. In summary, phellopterin, a novel component isolated from Changii radix, inhibited Ca²⁺ influx induced by stimulation of voltage‐gated and receptor‐dependent calcium channels with a greater inhibition of receptor‐dependent calcium channels. It also inhibited Ca²⁺ release from caffeine‐ryanodine and InsP₃‐sensitive internal stores, being more potent for caffeine stimulation. Phellopterin may be a promising candidate for the development of new classes of calcium antagonists. Drug Dev Res 68:79–83, 2007. © 2007 Wiley‐Liss, Inc.     

Characteristics of palytoxin-induced cation currents and Ca2+ mobilization in smooth muscle cells of rabbit portal vein

We examined the nature of the palytoxin (PTX)-induced channel and its relevance to the Ca²⁺ mobilizing effect of the toxin on smooth muscle cells isolated from rabbit portal vein using whole-cell voltage-clamp and microfluorimetric techniques. PTX (1 nM) induced a sustained, irreversible inward current at a holding potential of –40 mV. The PTX-induced current reversed at 0.5 ± 0.6 mV, and the PTX-induced channel permitted the passage of Na⁺, K⁺, Cs⁺ and, to a lesser extent, Li⁺, but not choline⁺ or Ca²⁺. During the sustained phase of the current, superfusion of Ni²⁺ (5 mM), La³⁺ (0.5 mM) or 2,4-dichlorobenzamil (2,4-DCB, 25 μM) reduced the current amplitude and decreased the slope conductance without changing the reversal potential. In 5 of 7 experiments, ouabain transiently increased the PTX-induced inward current and shifted the reversal potential in a positive direction. Subsequently, ouabain inhibited the current in every cell. PTX (10 nM) induced a sustained rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was resistant to verapamil but suppressed by omission of extracellular Ca²⁺. When external Na⁺ was replaced by choline⁺, PTX did not increase [Ca²⁺]_i. Pretreatment with 2,4-DCB prevented the elevation of [Ca²⁺]_i due to PTX. These results suggest that PTX does not directly stimulate Ca²⁺ entry but induces entry through Na⁺-Ca²⁺ exchange as a consequence of increased cytosolic Na⁺. Ni²⁺, La³⁺, 2,4-DCB and ouabain were shown to act as blockers of the PTX-induced channel. Ouabain may also inhibit Na⁺ pump current activated by cytosolic Na⁺. Received: 15 May 1996 / Accepted: 28 August 1996     

Neuropeptide Y as a stimulator of Na+-dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes

Several physiological stimuli cause a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiomyocytes. This increased [Ca²⁺]_i must be restored to physiological resting level to ensure response to further stimuli. In the present study, we examined the effect of neuropeptide Y (NPY), which is secreted from certain adrenergic or non-adrenergic neurons, on Ca²⁺ efflux from freshly isolated, quiescent adult rat cardiomyocytes. The isolated cardiomyocytes were preloaded with ⁴⁵CaCl₂ for 1 h. Then, the fractional release of ⁴⁵Ca²⁺ from the cells was measured. NPY stimulated the efflux of ⁴⁵Ca²⁺ from isolated adult rat cardiomyocytes in a concentration-dependent manner (10^–8 M to 10^–6 M). NPY (10^–6 M)-induced Ca²⁺ efflux was 2.0 ± 0.16% of the total cellular content. The ⁴⁵Ca²⁺ efflux from the cells was also stimulated by Y₁ receptor agonist, [Leu³¹, Pro³⁴]NPY, but not by Y₂ receptor agonist, NPY_13–36. The effect of NPY was inhibited by a peptide NPY inhibitor, NPY_18–36 and a non-peptide NPY inhibitor, benextramine to a similar extent. From these results, it is conceivable that the effect of NPY on Ca²⁺ efflux from cardiomyocytes is mediated through Y₁ receptors. It was also observed that NPY caused a rise in [Ca²⁺]_i to almost 150 nM. NPY-stimulated ⁴⁵Ca²⁺ efflux was not affected by removal of extracellular Ca²⁺, but was dependent on the presence of extracellular Na⁺. Moreover, NPY caused a ²²Na⁺ influx into the cells of about 1.6-fold over the basal value which was inhibited by amiloride and 5-(N,N-dimethyl)-amiloride, known Na⁺/Ca²⁺ exchange inhibitors. In addition, isoproterenol also caused ⁴⁵Ca²⁺ efflux from the cells and which was enhanced by the addition of NPY. These results suggest that NPY stimulates extracellular Na⁺-dependent ⁴⁵Ca²⁺ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane Y₁ receptors with which NPY may couple during Na⁺/Ca²⁺ exchange. Received: 21 May 1997 / Accepted: 26 August 1997     

An active metabolite of carbamazepine, carbamazepine-10,11-epoxide, inhibits ion channel-mediated catecholamine secretion in cultured bovine adrenal medullary cells

 We have recently reported inhibitory effects of carbamazepine (CBZ) on ion channel-mediated secretion of catecholamines in bovine adrenal medullary cells. Here, we report the effects of carbamazepine-10,11-epoxide (CBZ-E), an active metabolite of CBZ, and carbamazepine-10,11-diol (CBZ-D), a non-active metabolite, on ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured adrenal medullary cells. CBZ-E, but not CBZ-D inhibited ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion induced by carbachol or veratridine with a half-maximal inhibitory concentration (IC₅₀) of 0.26 or 0.68 μg/ml, respectively. CBZ-E also inhibited high K⁺-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion (IC₅₀ = 0.3 μg/ml), but CBZ-D did not. These findings suggest that CBZ-E, but not CBZ-D, attenuates catecholamine secretion by inhibiting nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na⁺ channels and voltage-dependent Ca²⁺ channels in the cells. This inhibition of CBZ-E as well as CBZ may be related to the clinical effects in neuropsychiatric disorders. Received: 13 May 1997 /Final version: 4 August 1997     

The Effect of High Calcium Intake on Intracellular Free [Ca2+] and Na+-H+ Exchange in DOC-NaCl-Hypertensive Rats

Abstract: The effects of calcium supplementation on blood pressure, intracellular free calcium concentration ([Ca²⁺],) and rate of Na⁺-H⁺ exchange were studied in DOC-NaCl-hypertensive rats. All the animals were uninephrectomized and divided into two main groups: the first group received deoxycorticosterone (DOC) (25 mg/kg, s.c.) once a week and had 0.7% NaCl as drinking fluid while the other received equal volumes of saline and tap water to drink. The animals were further divided according to dietary calcium intake: in the Control and DOC groups the chow contained 1.1% calcium, in the Calcium and DOC + Calcium groups, 2.5%. After 6 and 8 weeks, blood pressure in the DOC group was higher than in the Control group; on the other hand, the development of hypertension was attenuated in the DOC + Calcium compared with the DOC group. The Control and Calcium groups did not differ from each other. Platelets and lymphocytes were used as experimental models to study changes in the regulation of [Ca²⁺]ⁱ, evaluated by fluorescent indicators indo-1 and quin-2. In lymphocytes, basal [Ca²⁺]_i was highest in the DOC group, but similar in DOC + Calcium and Control groups. In platelets, both basal and thrombin-stimulated [Ca²⁺]_i were higher in the DOC and DOC + Calcium groups than in the Control group. In both cell types [Ca²⁺]_i was similar in Control and Calcium groups. In addition, platelets were used to study the ability of the cells to recover from intracellular acidification by first blocking the Na⁺ -H⁺ exchange in a Na⁺-free medium and then restarting the exchange mechanism by increasing the extracellular Na⁺ concentration at constant speed. Changes in [pH]_i were monitored by fluorescent indicator BCECF. The rate of Na⁺-H⁺ exchange in the recovery phase did not reveal differences between the experimental groups. Vascular smooth muscle function was examined by determining concentration-response curves for noradrenaline in mesenteric arterial rings and a shift to the left was observed in the DOC and DOC + Calcium groups compared with the Control group. The present data indicate that high calcium intake attenuates the development of mineralocorticoid-salt hypertension. The DOC-induced elevation of blood pressure is associated with higher [Ca²⁺]_i in platelets and lymphocytes, evidencing disturbances in cellular calcium handling. However, possibly due to differing characteristics in regulatory mechanisms between these cell types, supplementary calcium reduced [Ca²⁺]_i only in lymphocytes. The assumed elevation in intracellular sodium did not cause detectable changes in the Na⁺-H⁺ exchange rate in platelets. Enhanced vascular responses to noradrenaline observed in the DOC group were not altered by high calcium intake.     

Cellular Ca2+ Dynamics in Urinary Bladder Smooth Muscle From Transgenic Mice Overexpressing Na+-Ca2+ Exchanger

The rise of Ca²⁺ concentration ([Ca²⁺] _i ) by reducing external Na⁺ in urinary bladder smooth muscle cells (UBSMCs) from transgenic mice overexpressing Na⁺/Ca²⁺ exchanger type-1.3 (NCX1.3^tg/tg) was about 4 times as large as that in the wild-type (WT). NCX1 protein expression in UB increased about 4-fold in NCX1.3^tg/tg. The Ca²⁺ release by caffeine in UBSMCs was comparable between NCX1.3^tg/tg and WT, but [Ca²⁺]_i decay was faster in NCX1.3^tg/tg. Contractions induced by acetylcholine, 60 mM K⁺, or electrical stimulation were significantly smaller in UB segments of NCX1.3^tg/tg. NCX worked in Ca²⁺-extrusion mode during these contractions in UBSMCs of both WT and NCX1.3^tg/tg.     

Neomycin blocks dihydropyridine-insensitive Ca2+ influx in bovine adrenal chromaffin cells

There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by w-conotoxin-sensitive and -insensitive Ca²⁺ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 μM) and neomycin (0.5 mM) reduced high K⁺ (50 mM)-evoked intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients to a larger extent than each drug separately. [Ca²⁺]_i was measured using the fluorescent intracellular Ca²⁺ indicator fura-2. Neomycin (0.05−2 mM) reduced high K⁺-evoked ⁴⁵Ca²⁺ uptake in a dose-dependent manner (IC₅₀ = 0.09 mM). In the presence of nitrendipine (1 μM), the minimal neomycin concentration necessary for total blockade of ⁴⁵Ca²⁺ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the ⁴⁵Ca²⁺ uptake remaining in 0.3 mM neomycin (26% of maximum) was similar to the fractional inhibition by nitrendipine alone (29%). Neomycin (0.05−2 mM) inhibited the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1 μM) much more extensively at 2 mM than at 0.3 mM (percent inhibition = 59% and 15%, respectively). Neomycin (0.05−2 mM) blocked high K⁺-evoked noradrenaline and adrenaline release in a dose-dependent fashion (IC₅₀ = 0.8−1.1 mM), the blockade efficiency being enhanced in the presence of 1 μM nitrendipine (IC₅₀ = 0.17−0.19 mM). It is concluded that neomycin (≤ 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca²⁺ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca²⁺ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.     

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 μM evoked a robust [Ca²⁺]_i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 μM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca²⁺ medium. Removal of extracellular Ca²⁺ reduced the Ca²⁺ signals evoked by 50–100 μM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca²⁺ release and also extracellular Ca²⁺ influx. A concentration of 100 μM SKF 96365 caused significant Mn²⁺ quench of fura-2 fluorescence, which was partly inhibited by La³⁺ (1 mM) or Gd³⁺ (0.1 mM), indicating that the SKF 96365-induced Ca²⁺ influx had two components: one is sensitive to La³⁺ (1 mM) or Gd³⁺ (0.1 mM), the other is not. The internal Ca²⁺ source for the SKF 96365-induced [Ca²⁺]_i transient was the endoplasmic reticulum Ca²⁺ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca²⁺ pump nearly abolished the SKF 96365-induced [Ca²⁺]_i increase in Ca²⁺-free medium. In contrast, pretreatment with 100 μM SKF 96365 only partly depleted the thapsigargin-sensitive Ca²⁺ store. Addition of 10 mM Ca²⁺ induced a significant [Ca²⁺]_i increase after prior incubation with 100 μM SKF 96365 in Ca²⁺-free medium, demonstrating that SKF 96365 induced capacitative Ca²⁺ entry. This capacitative Ca²⁺ entry was about 40% of that induced by 1 μM thapsigargin. Additional to inducing its own capacitative Ca²⁺ entry, 100 μM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca²⁺ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca²⁺]_i transient. Inhibition of the plasma membrane Ca²⁺ pump with La³⁺ or Gd³⁺, or lowering extracellular Na⁺ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca²⁺]_i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca²⁺-free medium, the thapsigargin-induced [Ca²⁺]_i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca²⁺ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca²⁺ signaling. It induced a considerable increase in [Ca²⁺]_i by releasing Ca²⁺ from the endoplasmic reticulum store followed by capacitative Ca²⁺ entry. It also caused a direct Ca²⁺ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca²⁺ pump or Na⁺/Ca²⁺ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca²⁺ entry. Received: 21 September 1998 / Accepted: 2 December 1998     

Effect of the antidepressant maprotiline on calcium movement and the viability of renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of maprotiline, an antidepressant, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura-2. Maprotiline (>2.5 µM) caused a rapid rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ 200 µM). Maprotiline-induced [Ca²⁺]_i rise was reduced by removal of extracellular Ca²⁺ or by addition of La³⁺, but was not altered by voltage-gated Ca²⁺-channel blockers. Maprotiline-induced Mn²⁺ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca²⁺ influx. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, caused a monophasic [Ca²⁺]_i rise, after which the increasing effect of maprotiline on [Ca²⁺]_i was nearly abolished; also, pretreatment with maprotiline reduced a portion of thapsigargin-induced [Ca²⁺]_i rise. U73122, an inhibitor of phospholipase C, abolished [Ca²⁺]_i rise induced by ATP (but not by maprotiline). Overnight incubation with 1–10 µM maprotiline enhanced cell viability, but 20–50 µM maprotiline decreased it. These findings suggest that maprotiline rapidly increases [Ca²⁺]_i in renal tubular cells by stimulating both extracellular Ca²⁺ influx and intracellular Ca²⁺ release, and may modulate cell proliferation in a concentration-dependent manner.     

Effect of thymol on Ca homeostasis and viability in human glioblastoma cells

The effect of the natural essential oil thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human glioblastoma cells was examined. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Thymol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca²⁺]_i rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca²⁺]_i rise by inducing phospholipase C- and protein kinase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via non store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis.