Lack of Ca2+- and ATP-dependent priming stage in caffeine-induced exocytosis in bovine adrenal chromaffin cells: comparison with Ca2+

1. Caffeine (20-40 mM) secreted catecholamines from beta-escin-permeabilized bovine adrenal chromaffin cells in the presence or absence of 2 mM MgATP. The caffeine-induced catecholamine secretion in the presence of MgATP was to the same extent as that in the absence of MgATP. 2. Ca2+ (0.1-10 microM) induced a significantly greater secretion of catecholamines in the presence of MgATP than in the absence of MgATP. 3. ML-9 (100 microM) and ML-7 (100 microM), myosin light chain kinase inhibitors, and W-7 (100 microM) and trifluoperazine (TFP; 30 microM), calmodulin antagonists, inhibited the Ca2+-induced catecholamine secretion in the presence of MgATP but not in the absence of MgATP. They did not inhibit the caffeine-induced catecholamine secretion in the presence of MgATP. 4. The ATP-independent phase in Ca2+-dependent exocytosis is thought to be associated with the final step that ultimately leads to fusion, while the ATP-dependent phase is thought to be associated with a vesicle priming reaction. Therefore, these results suggest that the ATP-requiring priming stage is lacking in the process of caffeine-induced exocytosis in bovine adrenal chromaffin cells.     

Myosin light chain kinase inhibitors and calmodulin antagonists inhibit Ca2+– and ATP–dependent catecholamine secretion from bovine adrenal chromaffin cells

1 We have used stage-specific assays for ATP-dependent priming and for Ca²⁺-activated triggering in the absence of ATP to examine the effects of myosin light chain kinase (MLCK) inhibitors, ML-9 and ML-7, and calmodulin antagonists, W-7 and trifluoperazine (TFP), on regulated exocytosis in β-escin-permeabilized bovine adrenal chromaffin cells. 2 Ca²⁺ (0.1–30 μm ) induced a significantly greater secretion of catecholamines in the presence of MgATP (2 m m ) than in the absence of MgATP. 3 ML-9 (30 and 100 μm ), ML-7 (30 and 100 μm ), W-7 (30 and 100 μm ) and TFP (10 and 30 μm ) inhibited the Ca²⁺-induced catecholamine secretion in the presence of MgATP, but did not affect the catecholamine response to Ca²⁺ in the absence of MgATP. 4 In intact cells all these compounds inhibited catecholamine secretion in responses to acetylcholine (100 μm ) and high K⁺ (40 m m ). 5 The results obtained in permeabilized cells suggest that the calmodulin-MLCK system plays an essential role in the ATP-requiring priming stage but not in the Ca²⁺-triggered fusion step in the exocytotic process in bovine adrenal chromaffin cells.     

Histamine-induced Ca2+ release in bovine adrenal chromaffin cells

The histamine-induced biphasic increase of the intracellular free [Ca2+] ([Ca2+]i) was studied in bovine adrenal chromaffin cells using fura-2 microfluorimetry and the whole-cell patch-clamp technique. Both the rapid, transient Ca2+ rise and the sustained plateau component of elevated [Ca2+]i were independent of extracellular Ca2+. Incubation with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) blocker thapsigargin diminished histamine-induced changes in [Ca2+]i. When Ca2+ release was either stimulated by IP3 or blocked with the competitive inhibitor heparin, histamine was unable to elicit the typical Ca2+ rise. Ryanodine, tetracaine and ruthenium red, all blockers of Ca2+ release from caffeine-sensitive stores, had only minor effects on the agonist-induced Ca2+ changes. The contribution of mitochondria in shaping the histamine-induced Ca2+ increase was studied using ruthenium red and the two proton ionophores carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). Both mitochondrial uncouplers reversibly increased [Ca2+]i and induced an inward current leading to cell membrane depolarisation. In summary, these results indicate that Ca2+ from IP3-sensitive stores is essential for the generation of both the transient increase and secondary elevation in [Ca2+]i.     

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.     

Increase in catecholamine release and 45Ca2+ uptake induced by GABA in cultured bovine adrenal chromaffin cells

The role of Ca2+ in GABA-evoked catecholamine (CA) release from adrenal medulla was investigated in primary cultures of bovine adrenal chromaffin cells. GABA facilitated the 45Ca2+ uptake associated with the increase of Ca release in cultured bovine adrenal chromaffin cells. The effects of GABA on both 45Ca2+ uptake and CA release were blocked by bicuculline and picrotoxin. Nifedipine reduced the 45Ca2+ uptake and CA release induced by GABA. These data support our previous suggestion that the activation of GABA receptors on adrenal chromaffin cells facilitates the Ca2+ influx through voltage-sensitive Ca2+ channels, leading to the release of CA.     

Lysophospholipids elevate [Ca2+]i and trigger exocytosis in bovine chromaffin cells

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are responsible for many physiological functions, including angiogenesis, neuronal survival, and immunity. However, little is known about their effects in modulating the stimulus-secretion coupling in bovine chromaffin cells. The result of PCR showed that at least two receptors (S1P(3) and LPA(1)) were expressed in bovine chromaffin cells. The elevation of [Ca(2+)](i) by S1P was fast and sustaining; but the elevation by LPA was slow and transient. The EC(50) for S1P and LPA in elevating the [Ca(2+)](i) were 0.55+/-0.01 and 0.54+/-0.40microM, respectively. This elevation could be totally blocked by thapsigargin, 2-APB, and U73122. Pertussis toxin pretreatment inhibited about half of the elevation in [Ca(2+)](i) suggesting the involvement of G(i) and other G-proteins. Repetitive [Ca(2+)](i) elevations elicited by S1P, but not LPA, were inhibited by ryanodine. S1P was more effective than LPA in triggering exocytosis as measured by the changes in membrane capacitance. The whole-cell Ca(2+) current was inhibited by both lysophospholipids but Na(+) current was inhibited by S1P only. These results suggest the differential effects of LPA and S1P in releasing Ca(2+) from the intracellular Ca(2+) stores and modulating the stimulus-secretion coupling in bovine chromaffin cells.     

Amphetamine enhances Ca2+ entry and catecholamine release via nicotinic receptor activation in bovine adrenal chromaffin cells

Amphetamine, a psychostimulant, has been shown to act as a channel blocker of muscle nicotinic receptors and to induce a Ca(2+)-dependent secretion from adrenal chromaffin cells. In this study, the relationship between amphetamine and nicotinic receptors was studied using bovine adrenal chromaffin cells as a model system. Our results show that D-amphetamine sulfate alone induced an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [3H]norepinephrine release in a dose-dependent and extracellular Ca(2+)-dependent manner. Two common nicotinic receptor antagonists, hexamethonium and mecamylamine, suppressed the D-amphetamine sulfate-induced [Ca(2+)](c) rise and [3H]norepinephrine release. In addition, D-amphetamine sulfate inhibited the 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP)-induced [Ca(2+)](c) rise and [3H]norepinephrine release, but not the high K(+)- or veratridine-induced [Ca(2+)](c) increase and [3H]norepinephrine release. Antagonists, including alpha-bungarotoxin and choline, that are more specific for alpha7 nicotinic receptors were capable of inhibiting the D-amphetamine sulfate-induced [Ca(2+)](c) rise, while D-amphetamine sulfate was found to be capable of inhibiting the [Ca(2+)](c) rise induced by the alpha7-nicotinic receptor agonists, epibatidine and choline. Moreover, D-amphetamine sulfate dose-dependently suppressed [3H]nicotine binding to chromaffin cells. We, therefore, conclude that D-amphetamine sulfate acts as a nicotinic receptor agonist to induce [Ca(2+)](c) increase and [3H]norepinephrine release in bovine adrenal chromaffin cells.     

Differential requirements for Ca2+ concentrations for catecholamine release and biosynthesis in isolated bovine adrenal chromaffin cells

Differential effects of [Ca²⁺] on catecholamine release and biosynthesis in isolated bovine adrenal chromaffin cells were investigated. Carbamylcholine, an agonist of the nicotinic and muscarinic acetylcholine receptor, or Na⁺ deprivation in the incubation medium, stimulated catecholamine release and biosynthesis in these cells. The concentrations of extracellular [Ca²⁺] which stimulate catecholamine biosynthesis were less than those which stimulate catecholamine release. An increase in intracellular levels of free Ca²⁺ ([Ca²⁺]_i) induced by Na⁺ deprivation was dependent on extracellular [Ca²⁺]. These results indicate that, in bovine adrenal chromafn cells, catecholamine biosynthesis is regulated by lower levels of [Ca²⁺]_i than is catecholamine release.     

Failure of endothelin-1 to activate store-operated Ca2+ channels by lack of mobilization from intracellular Ca2+ stores in cultured bovine adrenal chromaffin cells

We have recently shown that in addition to L-type voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) stimulation opens two types of Ca2+-permeable nonselective cation channels [designated nonselective cation channel-1 (NSCC-1) and NSCC-2]. However, in this Ca2+ entry, the involvement of store-operated Ca2+ channel (SOCC), which is suggested to exist in chromaffin cells, was unclear. Those NSCCs as well as SOCC can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. To clarify whether SOCC should actually exist and play a role in Ca2+ entry in chromaffin cells stimulated with ET-1, we examined the effects of removal of extracellular Ca2+, thapsigargin (TG, an inhibitor of endoplasmic reticulum Ca2+-ATPase), LOE 908 and SK&F 96365 on cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured bovine adrenal chromaffin cells. After the cells were exposed to Ca2+-free medium followed by exposure to TG to deplete Ca2+ from the intracellular Ca2+ store, restoration of extracellular Ca2+ caused a gradual increase in [Ca2+]i (to about 200% of control). The increase was unaffected by LOE 908, but completely abolished by SK&F 96365. In the Ca2+-free medium, no increase in [Ca2+]i by ET-1 was observed, but the subsequent restoration of extracellular Ca2+ induced a rapid increase in [Ca2+]i (to the same level of [Ca2+]i as that evoked by ET-1 in the normal medium (1.0 mM Ca2+)). Since SK&F 96365 is also a blocker of SOCC, these results indicate that in bovine adrenal chromaffin cells, Ca2+ entry through SOCC (Ca2+ influx through the capacitative Ca2+ entry system) occurs but is comparably weak, and that it virtually does not work on the stimulation of ET-1.     

Pharmacological characterization of receptor-mediated Ca2+ entry in endothelin-1-induced catecholamine release from cultured bovine adrenal chromaffin cells

To clarify the mechanism for the endothelin-1 (ET-1)-induced release of catecholamines from the adrenal gland, we examined the effects of removal of extracellular Ca2+, blockers of L-, N-, P- and Q-types of voltage-operated Ca2+ channels (VOCC) such as nifedipine (L-type), omega-conotoxin GVIA (N-type), omega-agatoxin IVA (P-type) and omega-conotoxin MVIIC (Q-type) and blockers of voltage-independent Ca2+ entry channel such as SK&F 96365 and LOE 908 on release of catecholamines, the cytosolic free Ca2+ concentration ([Ca2+]i), and 45Ca2+ uptake in cultured bovine adrenal chromaffin cells. ET-1 but not ET-3 induced increases in release of catecholamines, [Ca2+]i, and 45Ca2+ uptake. The responses to ET-1 were abolished by the antagonist for ET(A) receptors, BQ-123, but not by the antagonist for ET(B) receptors, BQ-788, and they were abolished by removal of extracellular Ca2+. The increases were only partially inhibited (to about 65% of control) by nifedipine but unaffected by any of the omega-toxins. The nifedipine-resistant increase was inhibited by SK&F 96365 (to about 40%) and abolished by LOE 908 alone. These results indicate that ET-1 augments the release of catecholamines from adrenal chromaffin cells through ET(A) receptors, by activating two types of Ca2+ entry channels in addition to L-type VOCC: one (nonselective cation channel-1; NSCC-1) is sensitive to LOE 908 but resistant to SK&F 96365, whereas the other (NSCC-2) is sensitive to both LOE 908 and SK&F 96365.     

Inhibition by selenium compounds of catecholamine secretion due to inhibition of Ca2+ influx in cultured bovine adrenal chromaffin cells

Selenium is an essential trace metal element, whereas large doses of selenium exert adverse effects to the human body. We examined the effects of selenium compounds, sodium selenite (Na2SeO3) and sodium selenate (Na2SeO4), on catecholamine secretion from cultured bovine adrenal chromaffin cells. Treatment of chromaffin cells with sodium selenite for 72, 48, and 24 h caused decreases in protein and catecholamine contents, in association with cell damage, at concentrations over 30, 300, and 300 microM, respectively. The cells treated with subtoxic conditions (<100 microM, 48 h) of sodium selenite were used for further experiments. Sodium selenite treatment for 48 h inhibited carbachol (CCh)-induced catecholamine secretion in a concentration-dependent and non-competitive manner, while it did not affect high K+- and veratridine-induced catecholamine secretion. Sodium selenite (100 microM) did not affect CCh- and veratridine-induced 22Na+ influx, while the compound inhibited 45Ca2+ influx induced only by CCh, but not high K+ and veratridine. Sodium selenate even at higher concentrations (1000 microM) did not affect any stimulus-induced catecholamine secretion and 45Ca2+ influx. Thus, sodium selenite may specifically exert adverse effects, such as inhibition of physiological stimulus-induced catecholamine secretion from adrenal chromaffin cells due to inhibition of Ca2+ influx.     

L‐type Ca2+ channel opener BayK 8644‐induced Ca2+ influx and Ca2+ release in human oral cancer cells (OC2)

The effect of BayK 8644, a chemical widely used to activate L‐type Ca²⁺ channels, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human oral cancer cells (OC2) has not been explored to date. The present study examined whether BayK 8644 altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura‐2. BayK 8644 (10 pM–10 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. BayK 8644‐induced Ca²⁺ influx was blocked by nifedipine, but was not altered by the store‐operated Ca²⁺ entry inhibitors, econazole and SKF96365; protein kinase C modulators phorbol 12‐myristate 13‐acetate (PMA) and GF109203X; the protein kinase A inhibitor H89; and the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺‐free medium, after pretreatment with 1 µM BayK 8644, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pretreatment abolished BayK 8644‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change BayK 8644‐induced [Ca²⁺]_i rises. Collectively, in OC2 cells, BayK 8644 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via L‐type Ca²⁺ channels. Drug Dev Res 69: 2008. © 2008 Wiley‐Liss, Inc.     

In vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules: Specific control by Ca2+

Summary The control of the in vitro interaction between bovine adrenal medullary plasma membernes and chromaffin granules by calcium has been studied. This interaction, which has previously been shown to result in the release of the soluble granular content, is a possible cell-free model for exocytosis. The plasma memberane-induced catecholamine release was stimulated when the [Ca²⁺] exceeded 2×10^–7M. A maximal release was reached at 10^–5M with a half maximal response around 10^–6M. Mg²⁺ was not able to stimulate the system in the absence of Ca²⁺. These data suggest a high specificity of the calcium controlled exocytotic mechanism and the absence of an antagonism by high concentrations (10^–5–10^–1M) of magnesium at the exocytotic site itself.     

Inhibition by SEA0400, a selective inhibitor of Na+/Ca2+ exchanger, of Na+ -dependent Ca2+ uptake and catecholamine release in bovine adrenal chromaffin cells

The effects of SEA0400, a selective inhibitor of the Na(+)/Ca(2+) exchanger (NCX), on Na(+)-dependent Ca(2+) uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na(+) by treatment with ouabain and veratridine. SEA0400 inhibited Na(+)-dependent (45)Ca(2+) uptake and CA release, with the IC(50) values of 40 and 100 nM, respectively. The IC(50) values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 microM, respectively. These results indicate that SEA0400 is about 40 times more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine and DMPP. The IC(50) values of SEA0400 were 5.1 and 4.5 microM and the values of KB-R7943 were 2.6 and 2.1 microM against the release induced by acetylcholine and DMPP, respectively, indicating that the potency of SEA0400 is about a half of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release. The binding of [(3)H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC(50) = 90 microM) and KB-R7943 (IC(50) = 12 microM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.     

Role of Ca2+/phospholipid-dependent protein kinase in catecholamine secretion from bovine adrenal medullary chromaffin cells

The role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in catecholamine secretion from bovine adrenal medullary chromaffin cells was examined using four protein kinase C inhibitors: polymyxin B, sphingosine, staurosporine, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). For this purpose, digitonin-permeabilized chromaffin cells were used. Secretion of catecholamines from these cells was stimulated by the addition of micromolar amounts of exogenous free Ca2+. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and arachidonic acid, activators of protein kinase C, enhanced the catecholamine secretion evoked by Ca2+. But phorbol-12, 13-diacetate, a phorbol ester analog that does not activate protein kinase C, had no effect on Ca2(+)-evoked secretion. Polymyxin B at a low concentration (1 microM) abolished the enhancement of secretion by TPA or arachidonic acid without affecting the secretion evoked by Ca2+. However, polymyxin B at higher concentrations (10-100 microM) greatly reduced Ca2+-evoked catecholamine secretion. Sphingosine 10 microM-1 mM), Staurosporine (100 nM-1 microM, and H-7 (100-500 microM) inhibited TPA- or arachidonic acid-enhanced secretion but not Ca2(+)-evoked secretion. In cells in which protein kinase C was down-regulated by TPA, specific binding of [3H]phorbol-12,13-dibutyrate to the cells almost disappeared and the enhancement of secretion by TPA was no longer observed, whereas Ca2(+)-evoked secretion was maintained. These results strongly suggest that protein kinase C is not essential for the Ca2(+)-dependent catecholamine secretion from bovine adrenal chromaffin cells, but acts instead as a modulator.     

Antimigraine dotarizine blocks P/Q Ca2+ channels and exocytosis in a voltage-dependent manner in chromaffin cells

The mechanism of blockade of P/Q Ca(2+) channels by antimigraine, dotarizine, was studied in voltage-clamped bovine adrenal chromaffin cells. Inward currents through P/Q channels were pharmacologically isolated by superfusing the cells with omega-conotoxin GVIA (1 microM) plus nifedipine (3 microM). Dotarizine (10-30 microM) blocked the P/Q fraction of I(Ba) and promoted current inactivation. Thus, dotarizine caused a greater blockade of the late I(Ba), compared with blockade of the early peak I(Ba). This effect was more prominent, the longer was the duration of the depolarising pulse. The blockade of I(Ba) was also greater at more depolarising holding potentials (i.e. -60 mV), than was the blockade produced at more hyperpolarising holding potentials (i.e. -80 or -110 mV). Catecholamine secretory responses to brief pulses (2 s) of a Krebs-HEPES solution containing 100 mM K(+) and 2 mM Ca(2+) was blocked by 3 microM dotarizine. Blockade was faster and greater when dotarizine was applied on cells that were previously depolarised with Krebs-HEPES deprived of Ca(2+) and containing increasing concentrations of K(+). This voltage-dependent blockade of P/Q channels and exocytosis might be the underlying mechanism explaining the dotarizine prophylaxis of migraine attacks.     

Cyclic ADP-ribose requires FK506-binding protein to regulate intracellular Ca2+ dynamics and catecholamine release in acetylcholine-stimulated bovine adrenal chromaffin cells

The present study was undertaken to elucidate whether cyclic ADP-ribose (cADPR) mediates the amplification of Ca2+ signaling and catecholamine release via the involvement of FK506-binding proteins (FKBPs)/ryanodine receptor (RyR) in bovine adrenal chromaffin cells. cADPR induced Ca2+ release in digitonin-permeabilized chromaffin cells and this was blocked by FK506 and rapamycin, ligands for FKBPs; 8Br-cADPR, a competitive antagonist for cADPR; and antibody for FKBP12/12.6, while it was enhanced by cyclosporin A. Ryanodine-induced Ca2+ release was not affected by 8Br-cADPR and was remarkably enhanced by FK506, rapamycin, cyclosporin A, and cADPR. FK506 binds to FKBP12.6 and removes it from RyRs, but cADPR did not affect the binding between FKBP12.6 and RyR. In intact chromaffin cells, 8Br-cADPR, FK506, and rapamycin, but not cyclosporin A attenuated the sustained intracellular free Ca2+ concentration ([Ca2+]i) rise induced by acetylcholine (ACh). 8Br-cADPR, FK506, and SK&F 96365 reduced the Mn2+ entry stimulated with ACh only when Ca2+ was present in the extracellular medium. 8Br-cADPR, FK506, and rapamycin concentration-dependently inhibited the ACh-induced catecholamine (CA) release. Here, we present evidence that FKBP12.6 associated with RyR may be required for Ca2+ release induced by cADPR in bovine adrenal chromaffin cells. cADPR-mediated Ca2+ release from endoplasmic reticulum in ACh-stimulated chromaffin cells is coupled with Ca2+ influx through the plasma membrane which is essential for ACh-stimulated CA release.     

Ca2+ mobilization induced by δ‐hexachlorocyclohexane in Madin Darby canine kidney cells

The effect of the pesticide δ‐hexachlorocyclohexane (δ‐HCH) were examined on Ca²⁺ signaling in Madin Darby canine kidney (MDCK) using fura‐2 as a Ca²⁺ probe. δ‐HCH at concentrations of 5–200 mM increased intracellular free Ca²⁺ concentration ([Ca²⁺]_i) concentration‐dependently. The [Ca²⁺]_i increase comprised an immediate rise followed by a sustained phase within 5 min of measurement. External Ca²⁺ removal slightly reduced the [Ca²⁺]i increase. In Ca²⁺‐free medium, 150 μM δ‐HCH did not increase [Ca²⁺]_i after pretreatment with carbonylcyanide m‐chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and two endoplasmic reticulum (ER) Ca²⁺ pump inhibitors, thapsigargin (1 μM) and cyclopiazonic acid (100 μM). Conversely, pretreatment with δ‐HCH prevented thapsigargin, cyclopiazonic acid, and CCCP from releasing more Ca²⁺, suggesting 150 μM δ‐HCH released Ca²⁺ from the ER and mitochondria. δ‐HCH (150 μM) activated Mn²⁺ quench of fura‐2 fluorescence, confirming that δ‐HCH induced Ca²⁺ influx. Addition of 3 mM Ca²⁺ induced a concentration‐dependent [Ca²⁺]_i increase after pretreatment with 100–200 μM δ‐HCH for 870 sec in Ca²⁺‐free medium. The δ‐HCH (150 μM)‐induced Ca²⁺ release was decreased by inhibiting phospholipase C with 1 μM U73122. Collectively, we have found that δ‐HCH increased [Ca²⁺]_i in MDCK cells by releasing Ca²⁺ from the ER and mitochondria, followed by capacitative Ca²⁺ entry. Drug Dev. Res. 50:186–192, 2000. © 2000 Wiley‐Liss, Inc.     

Maprotiline‐induced Ca2+ fluxes and apoptosis in human osteosarcoma cells

The effect of maprotiline on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in human osteosarcoma cells (MG63), using the fluorescent dyes fura‐2 and WST‐1, respectively. Maprotiline at concentrations of ≥20 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca²⁺. The maprotiline‐induced Ca²⁺ influx was sensitive to inhibition by aristolochic acid (a phospholipase A₂ inhibitor). In Ca²⁺‐free medium, after treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), 200 µM maprotiline failed to induce a [Ca²⁺]_i rise. At concentrations of 50–100 µM maprotiline killed cells in a concentration‐dependent manner. The cytotoxic effect of 60 µM maprotiline was slightly enhanced by prechelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Propidium iodide staining data suggested that maprotiline induced apoptosis between concentrations of 60–70 µM, which was enhanced by BAPTA. Collectively, in MG63 cells, maprotiline induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from phospholipase A₂‐regulated Ca²⁺ channels. Furthermore, maprotiline caused apoptosis that was regulated by Ca²⁺. Drug Dev Res 71: 268–274, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of a novel, hydrophilic dihydropyridine, NKY-722, as a Ca2+ antagonist in bovine cultured adrenal chromaffin cells.

1. To characterize NKY-722, a novel hydrophilic dihydropyridine derivative, as a Ca2+ antagonist, we examined its effects on 45Ca2+ influx, intracellular free Ca2+ concentrations [( Ca2+]i), and release of noradrenaline and adrenaline in bovine cultured adrenal chromaffin cells. 2. NKY-722 had little effect on basal 45Ca2+ influx into the resting cells, but inhibited high K+ (35.9 mM)-evoked 45Ca2+ influx in a concentration-dependent manner with an IC50 value of 5.2 nM. 3. NKY-722 inhibited high K(+)-evoked increases in [Ca2+]i in a concentration-dependent manner without effect on the resting [Ca2+]i. 4. NKY-722 had little effect on basal release of noradrenaline and adrenaline but inhibited high K(+)-evoked release of noradrenaline and adrenaline in a concentration-dependent manner with IC50 values of 5.0 nM and 4.8 nM, respectively. 5. Nicardipine, a prototype of NKY-722, also inhibited high K(+)-evoked 45Ca2+ influx and release of noradrenaline and adrenaline in a concentration-dependent manner: the IC50 value for high K(+)-evoked 45Ca2+ influx was 51 nM, and the values for high K(+)-evoked release of noradrenaline and adrenaline were 52 nM and 50 nM, respectively. 6. These results show that NKY-722 is a hydrophilic Ca2+ antagonist ten times more potent than nicardipine.