Cotinine inhibits catecholamine release evoked by cholinergic stimulation from the rat adrenal medulla

The aim of the present study was to clarify whether cotinine affects the release of catecholamines (CA) from the isolated perfused rat adrenal gland, and to establish the mechanism of its action, in comparison with the response of nicotine. Cotinine (0.3-3 mM), when perfused into an adrenal vein for 60 min, inhibited CA secretory responses evoked by ACh (5.32 mM), DMPP (a selective neuronal nicotinic agonist, 100 microM for 2 min) and McN-A-343 (a selective muscarinic M1-agonist, 100 microM for 2 min) in dose- and time-dependent manners. However, cotinine did not affect CA secretion by high K+ (56 mM). Cotinine itself also failed to affect basal CA output. Furthermore, in the presence of cotinine (1 mM), CA secretory responses evoked by Bay-K-8644 (an activator of L-type Ca2+ channels, 10 microM) and cyclopiazonic acid (an inhibitor of cytoplasmic Ca2+-ATPase, 10 microM) were relative time-dependently attenuated. However, nicotine (30 microM), given into the adrenal gland for 60 min, initially rather enhanced CA secretory responses evoked by ACh and high K+, followed by the inhibition later, while it time-dependently depressed the CA release evoked by McN-A-343 and DMPP. Taken together, these results suggest that cotinine inhibits greatly CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, but does fail to affect that by the direct membrane-depolarization. It seems that this inhibitory effect of cotinine may be exerted by the cholinergic blockade, which is associated with blocking both the calcium influx into the rat adrenal medullary chromaffin cells and Ca2+ release from the cytoplasmic calcium store. It also seems that there is a big difference in the mode of action between cotinine and nicotine in the rat adrenomedullary CA secretion.     

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.     

Inhibition of Nicotinic Responses by Cotinine in Bovine Adrenal Chromaffin Cells

Abstract: We studied the effects of cotinine, the major metabolite of nicotine, on nicotine-induced increase in [³H]phorbol dibutyrate binding, activation of protein kinase C and [³H]noradrenaline release in primary cultured bovine adrenal chromaffin cells. Cotinine (1 mM, 15 min.) and nicotine (10 μM, 5 min.) increased the [³H]phorbol binding by 100% and 150%, respectively. Both a short-term (10 min.) and a long-term (24 hr) pretreatment with cotinine inhibited the effect of nicotine. A 24 hr pretreatment with cotinine (1 mM) also reduced the nicotine-induced increase in membrane-bound protein kinase C activity. Cotinine pretreatment (10 min.) dose-dependently inhibited the release of [³H]noradrenaline induced by nicotine and dimethylphenylpiperazinium. Cotinine pretreatment did not reduce the [³H]noradrenaline release induced by high extracellular potassium (56 mM) or veratrine (10 mg l^–1)- The results indicate that cotinine inhibits activation of protein kinase C and noradrenaline release induced by nicotinic agonists in primary cultures of bovine adrenal chromaffin cells. The results suggest that pre-existing cotinine could modify responses to acute exposure to nicotine in neural systems.     

Inhibitory effects of tramadol on nicotinic acetylcholine receptors in adrenal chromaffin cells and in Xenopus oocytes expressing alpha 7 receptors

1. Tramadol has been used clinically as an analgesic; however, the mechanism of its analgesic effects is still unknown. 2. We used bovine adrenal chromaffin cells to investigate effects of tramadol on catecholamine secretion, nicotine-induced cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases and membrane current changes. We also investigated effects of tramadol on alpha7 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes. 3. Tramadol concentration-dependently suppressed carbachol-induced catecholamine secretion to 60% and 27% of the control at the concentration of 10 and 100 microM, respectively, whereas it had little effect on veratridine- or high K(+)-induced catecholamine secretion. 4. Tramadol also suppressed nicotine-induced ([Ca(2+)](i)) increases in a concentration-dependent manner. Tramadol inhibited nicotine-induced inward currents, and the inhibition was unaffected by the opioid receptor antagonist naloxone. 5. Tramadol inhibited nicotinic currents carried by alpha7 receptors expressed in Xenopus oocytes. 6. Tramadol inhibited both alpha-bungarotoxin-sensitive and -insensitive nicotinic currents in bovine adrenal chromaffin cells. 7. In conclusion, tramadol inhibits catecholamine secretion partly by inhibiting nicotinic AChR functions in a naloxone-insensitive manner and alpha7 receptors are one of those inhibited by tramadol.     

Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons

The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and membrane currents were investigated in neonatal rat intracardiac neurons.In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca(2+)](I), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca(2+)](i) transients was 28 microM, close to the estimated clinical EC(50) (clinically relevant (half-maximal) effective concentration) of thiopental. In fura-2-loaded neurons, voltage clamped at -60 mV to eliminate any contribution of voltage-gated Ca(2+) channels, thiopental (25 microM) simultaneously inhibited nAChR-induced increases in [Ca(2+)](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by approximately 40% at -120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC(50) were also shown to inhibit nAChR-induced increases in [Ca(2+)](i) by approximately 40%.Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca(2+)](i), indicating that inhibition of Ca(2+) release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. Depolarization-activated Ca(2+) channel currents were unaffected in the presence of thiopental (25 microM), pentobarbital (50 microM) and ketamine (10 microM). In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca(2+)](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions.     

The novel Na(+)/Ca(2+) exchange inhibitor KB-R7943 also blocks native and expressed neuronal nicotinic receptors

We studied the effects of the novel Na(+)/Ca(2+) exchange inhibitor KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (AChRs) expressed in XENOPUS: oocytes. As expected, KB-R7943 blocked the Na(+)-gradient dependent (45)Ca(2+) uptake into chromaffin cells (IC(50) of 5.5 microM); but in addition, the compound also inhibited the (45)Ca(2+) entry and the increase of cytosolic Ca(2+) concentration, [Ca(2+)](c), stimulated by 5 s pulses of ACh (IC(50) of 6.5 and 1.7 microM, respectively). In oocytes expressing alpha(3)beta(4) and alpha(7) nicotinic AChRs, voltage-clamped at -60 mV, inward currents elicited by 1 s pulses of 100 microM ACh (I(ACh)) were blocked by KB-R7943 with an IC(50) of 0.4 microM and a Hill coefficient of 0.9. Blockade of alpha(3)beta(4) currents by KB-R7943 was noncompetitive; moreover, the blocker (0.3 microM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 microM and 1 mM ACh, respectively). Inhibition of alpha(3)beta(4) currents by 0.3 microM KB-R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 microM), the inhibition amounted to 33, 64 and 80% in oocytes voltage-clamped at -40, -60 and -100 mV, respectively. The onset of blockade was faster and the recovery slower at -100 mV; the reverse was true at -40 mV. In conclusion, KB-R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open-channel blocker at the rat brain alpha(3)beta(4) AChR. These results should be considered when KB-R7943 is to be used to study Ca(2+) homeostasis in cells expressing nicotinic AChRs and the Na(+)/Ca(2+) exchanger.     

Palytoxin-induced increase in cytosolic-free Ca(2+) in mouse spleen cells

The effect of palytoxin (C(129)H(223)N(3)O(54)) on Ca(2+) homeostasis in immune cells has not been studied. Therefore, we investigated the effect of palytoxin on the cytosolic-free Ca(2+) concentration ([Ca(2+)](i)) in mouse spleen cells using a fluorescence Ca(2+) indicator, fura-2. Palytoxin (0.1-100 nM) increased [Ca(2+)](i) in a concentration-dependent manner. The palytoxin-induced increase in [Ca(2+)](i) was abolished by the omission of extracellular Ca(2+) or 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100 microM), and was greatly inhibited by Ni(2+) (2 mM). Ouabain (0.5-1 mM) partially inhibited the palytoxin-induced response. There was no effect of decreased extracellular Na(+) (6.2 mM), tetrodotoxin (1 microM), verapamil (10 microM), nifedipine (10 microM), omega-agatoxin IVA (200 nM), omega-conotoxin GVIA (1 microM), omega-conotoxin MVIIC (500 nM), or La(3+) (100 microM). These results suggest that palytoxin increases [Ca(2+)](i) in mouse spleen cells by stimulating Ca(2+) entry through an SKF-96365-, Ni(2+)-sensitive pathway.     

Mesaconitine-induced relaxation in rat aorta: role of Na+/Ca2+ exchangers in endothelial cells

Previously, we reported that mesaconitine, an aconite alkaloid, increased intracellular Ca(2+) concentration ([Ca(2+)](i)) level in endothelium and caused relaxation in rat aorta via nitric oxide production. In the present study, we investigated the mechanisms of increase in the [Ca(2+)](i) level induced by mesaconitine in rat aorta and in human umbilical vein endothelial cells (HUVECs). Treatment with the low Na(+) buffer delayed the 30 microM mesaconitine-, but not 10 microM acetylcholine-, induced relaxation in rat aorta. Treatments with an inhibitor of Na(+)/Ca(2+) exchangers (20 microM 3',4'-dichlorobenzamil) and a reversed mode (Ca(2+) influx) inhibitor of the exchangers (30 microM 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate, KBR7943) showed similar effects. In HUVECs, 30 microM mesaconitine increased the [Ca(2+)](i) level in the presence of extracellular CaCl(2) and NaCl, and the response was inhibited by KBR7943. Mesaconitine increased intracellular Na(+) concentration level in HUVECs. The [Ca(2+)](i) response by mesaconitine was inhibited by 100 microM D-tubocurarine (an inhibitor of nicotinic acetylcholine receptors), but was not inhibited in the glucose-free buffer and by inhibitors of Na(+)/H(+) exchangers. These findings suggest that mesaconitine stimulated Ca(2+) influx via the Na(+)/Ca(2+) exchangers in endothelial cells and caused relaxation in the aorta. The possibility of D-tubocurarine-sensitive Na(+) channels as target(s) of mesaconitine is discussed.     

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.     

The mechanism underlying the contractile effect of a chemotactic peptide, formyl-Met-Leu-Phe on the guinea-pig Taenia coli

1 The contractile mechanism of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) was investigated in the guinea-pig Taenia coli, by simultaneously monitoring the changes in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) and force. 2 fMLP induced a significant elevation of [Ca(2+)](i) and force at concentrations higher than 10 nM. The maximal response was obtained at a concentration of higher than 1 microM. 3 fMLP (10 microM) augmented the force development induced by a stepwise increment of the extracellular Ca(2+) concentration during 60 mM K(+) depolarization, while it had no effect on the [Ca(2+)](i) elevation, and thus produced a greater force for a given elevation of [Ca(2+)](i) than 60 mM K(+) depolarization. 4 The removal of extracellular Ca(2+) completely abolished the fMLP-induced contraction. The fMLP-induced [Ca(2+)](i) elevation was inhibited substantially but not completely by 10 microM diltiazem, partly by 10 microM SK&F 96365, and completely by their combination. 5 Y27632, a specific inhibitor of rho-kinase, had no significant effect on the fMLP-induced [Ca(2+)](i) elevation and force development. 6 Chenodeoxycholic acid, a formyl peptide receptor antagonist, specifically abolished the fMLP-induced contraction but not high K(+)- or carbachol-induced contractions. 7 A dual lipoxygenase/cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, a nonselective leukotriene receptor antagonist, and a selective type 1 cysteinyl-containing leukotriene receptor antagonist specifically reduced the fMLP-induced contraction. 8 We suggest that the low-affinity-type fMLP receptor and lipoxygenase metabolites of arachidonic acid are involved in the fMLP-induced contraction in the guinea-pig T. coli. This contraction mainly depends on the [Ca(2+)](i) elevation due to Ca(2+) influx and the enhancement of Ca(2+) sensitivity in the contractile apparatus.     

Effects of chronic drug treatments on increases in intracellular calcium mediated by nicotinic acetylcholine receptors in SH-SY5Y cells

1. SH-SY5Y cells express alpha7 and alpha3* subtypes of nicotinic acetylcholine receptors (AChR). Numbers of these receptors are upregulated by chronic treatment with nicotinic agonists or KCl. In this study we have examined the functional consequences of these drug treatments on nicotine- or KCl-evoked increases in [Ca(2+)](i), in SH-SY5Y cells. 2. In untreated cells, nicotine increased [Ca(2+)](i) (EC(50) 7.5 microM). Responses to 10 microM nicotine were abolished by the non-selective nicotinic antagonist mecamylamine and were partially blocked by alpha7-selective antagonists, the alpha3beta2*-selective antagonist alpha-conotoxin-MII, and by cadmium and verapamil. 3. After treatment for 4 days with nicotinic agonists, nicotine-evoked increases in [Ca(2+)](i) were significantly decreased by about 25%. Nicotine-evoked responses were paradoxically increased in the presence of acute methyllycaconitine (MLA; an alpha7-selective antagonist) although other alpha7-selective antagonists were without effect, while alpha-conotoxin-MII gave a partial inhibition. The increase observed with MLA was abolished by mecamylamine but not by alpha-conotoxin-MII and was still observed 24 h after chronic nicotine treatment. 4. After treatment for 4 days with KCl, nicotine-evoked increases in [Ca(2+)](i) were also decreased by 25%, but acute MLA was without effect. Responses to 20 mM KCl were unchanged by prior treatment with nicotine or KCl. Treatment for 4 days with 5 microM verapamil reduced responses to both nicotine and KCl by about 50%. 5. Multiple nicotinic AChR subtypes contribute to nicotine-evoked increases in [Ca(2+)](i) in SH-SY5Y cells. Responses to acute nicotine are reduced after chronic nicotine or KCl treatment, with loss of the component attributed to the alpha7 subtype. However, in nicotine-treated cells this effect is reversed when nicotine stimulation is applied in the presence of acute MLA. The antagonist may assist in converting a non-functional alpha7 nicotinic AChR to a conducting state.     

Monensin causes transient calcium ion influx into mouse splenic lymphocytes in a sodium ion-independent fashion

Monensin, a Na(+) ionophore, can increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in many cell types, but no studies have investigated the mechanism underlying a monensin-induced increase in [Ca(2+)](i) in immune cells. In view of this, we investigated the effect of monensin on [Ca(2+)](i) and cytosolic free Na(+) concentration ([Na(+)](i)) in mouse splenic lymphocytes using a fluorescence Ca(2+) indicator, fura-2, and a fluorescence Na(+) indicator, sodium-binding benzofuran isophthalate (SBFI), respectively. Monensin (1-100 microM) caused transient and sustained increases in [Ca(2+)](i) and [Na(+)](i), respectively, in a concentration-dependent manner. The monensin-induced increase in [Ca(2+)](i) was abolished by the omission of extracellular Ca(2+) or 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100-150 microM), and was largely inhibited by Ni(2+) (2-5 mM). The omission of extracellular Na(+) failed to inhibit the monensin-induced increases in [Ca(2+)](i). Furthermore, tetrodotoxin (1-10 microM), 5-(N,N-dimethyl)-amiloride (DMA, 10-20 microM), 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400, 3-10 microM), verapamil (10-200 microM), nifedipine (10-200 microM), omega-agatoxin IVA (0.2-10 microM), omega-conotoxin GVIA (1-10 microM), omega-conotoxin MVIIC (0.5-10 microM), and nordihydroguaiaretic acid (NDGA, 1-10 microM) had no effect on the increases in [Ca(2+)](i). Monensin-induced Mn(2+) influx into splenic lymphocytes. The Mn(2+) influx was completely inhibited by SKF-96365. These results suggest that monensin transiently increases [Ca(2+)](i) in mouse splenic lymphocytes by stimulating Ca(2+) entry via non-selective cation channels in a Na(+)-independent manner.     

A novel action of the antianginal drug bepredil: induction of internal Ca(2+) release and external Ca(2+) influx in Madin-Darby canine kidney (MDCK) epithelial cells

The effect of the antianginal drug bepridil on Ca(2+) signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Bepridil at 10-50 microM evoked a significant rise in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in a dose-dependent manner. The [Ca(2+)](i) rise consisted of an immediate initial rise and a slow decay. Removal of external Ca(2+) partly inhibited the Ca(2+) signals by reducing both the initial rise and the decay phase, suggesting that bepridil activated both external Ca(2+) influx and internal Ca(2+) release. In Ca(2+)-free medium, pretreatment with 50 microM bepridil nearly abolished the Ca(2+) release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, and vice versa, pretreatment with thapsigargin inhibited most of the bepridil-induced Ca(2+) release, suggesting that the thapsigargin-sensitive Ca(2+) store was the main source of bepridil-induced Ca(2+) release. Bepridil (50 microM) induced considerable Mn(2+) quench of fura-2 fluorescence at an excitation wavelength of 360 nm, which was partly inhibited by La(3+) (0.1 mM). Consistently, La(3+) (0.1 mM) pretreatment significantly inhibited the bepridil-induced [Ca(2+)](i) rise. Addition of 3 mM Ca(2+) induced a significant [Ca(2+)](i) rise after prior incubation with 10-50 microM bepridil in Ca(2+)-free medium, suggesting that bepridil induced dose-dependent capacitative Ca(2+) entry. However, 50 microM bepridil inhibited 1 microM thapsigargin-induced capacitative Ca(2+) entry by 38%. Pretreatment with aristolochic acid (40 microM) so as to inhibit phospholipase A(2) inhibited 50 microM bepridil-induced internal Ca(2+) release by 42%, but inhibition of phospholipase C with U73122 (2 microM) or inhibition of phospholipase D with propranolol (0.1 mM) had little effect, suggesting that bepridil induced internal Ca(2+) release in an inositol 1,4,5-trisphosphate-independent manner that could be modulated by phospholipase A(2)-coupled events. This is the first report providing evidence that bepridil, currently used as an antianginal drug, induced a rise in [Ca(2+)](i) in a non-excitable cell line.     

Xestospongin C, a novel blocker of IP3 receptor, attenuates the increase in cytosolic calcium level and degranulation that is induced by antigen in RBL-2H3 mast cells

1. We evaluated the role of the cross-linking of Fc epsilon RI-mediated inositol 1,4,5-triphosphate (IP(3)) in the increase in cytosolic Ca(2+) level ([Ca(2+)](i)) using xestospongin C, a selective membrane permeable blocker of IP(3) receptor, in RBL-2H3 mast cells. 2. In the cells sensitized with anti-dinitrophenol (DNP) IgE, DNP-human serum albumin (DNP-HSA) and thapsigargin induced degranulation of beta-hexosaminidase and a sustained increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited both of these changes that were induced by DNP-HSA without changing those induced by thapsigargin. 3. In the absence of external Ca(2+), DNP-HSA induced a transient increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited this increase in [Ca(2+)](i). 4. In the cells permeabilized with beta-escin, the application of IP(3) decreased Ca(2+) in the endoplasmic reticulum (ER) as evaluated by mag-fura-2. Xestospongin C (3 - 10 microM) inhibited the effect of IP(3). 5. After the depletion of Ca(2+) stores due to stimulation with DNP-HSA or thapsigargin, the addition of Ca(2+) induced capacitative calcium entry (CCE). Xestospongin C (3 - 10 microM) inhibited the DNP-HSA-induced CCE, whereas it did not affect the thapsigargin-induced CCE. 6. These results suggest that Fc epsilon RI-mediated generation of IP(3) contributes to Ca(2+) release not only in the initial phase but also in the sustained phase of the increase in [Ca(2+)](i), resulting in prolonged Ca(2+) depletion in the ER. The ER Ca(2+) depletion may subsequently activate CCE to achieve a continuous [Ca(2+)](i) increase, which is necessary for degranulation in the RBL-2H3 mast cells. Xestospongin C may inhibit Ca(2+) release and consequently may attenuate degranulation.     

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.     

Effect of betulinic acid on intracellular-free Ca(2+) levels in Madin Darby canine kidney cells

The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca(2+) ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca(2+) dye. Betulinic acid caused significant increases in [Ca(2+)](i) concentration dependently between 25 and 500 nM with an EC(50) of 100 nM. The [Ca(2+)](i) signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca(2+) by 45+/-10%. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) abolished 250 microM betulinic acid-induced [Ca(2+)](i) increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 250 nM betulinic acid in Ca(2+)-free medium for 5 min. This [Ca(2+)](i) increase was not altered by the addition of 20 microM SKF96365 and 10 microM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) abolished 250 nM betulinic acid-induced Ca(2+) release. Pretreatment with 10 microM La(3+) inhibited 250 nM betulinic acid-induced [Ca(2+)](i) increases by 85+/-3%; whereas 10 microM of verapamil, nifedipine and diltiazem had no effect. In Ca(2+) medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 microM ATP and 1 microM thapsigargin-induced [Ca(2+)](i) increases by 33+/-3% and 45+/-3%, respectively. Trypan blue exclusion revealed that acute exposure of 250 nM betulinic acid for 2-30 min decreased cell viability by 6+/-2%, which could be prevented by pretreatment with 2 microM U731222. Together, the results suggest that betulinic acid induced significant [Ca(2+)](i) increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca(2+)](i) signal was contributed by an inositol 1,4, 5-trisphosphate-dependent release of intracellular Ca(2+) from thapsigargin-sensitive stores, and by inducing Ca(2+) entry from extracellular medium in a La(3+)-sensitive manner.     

Inhibition of nicotinic receptor-mediated catecholamine secretion by dryobalanops aromatica in bovine adrenal chromaffin cells

Effect of the aqueous extract from a medicinal plant Dryobalanops aromatica(Dipterocarpaceae) on catecholamine secretion was investigated in bovine adrenal chromaffin cells. The aqueous extract inhibited [(3)H]norepinephrine ([(3)H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, with a half-maximal inhibitory concentration (IC(50)) of 8.4 +/- 1.7 microgml(-1). Increases in cytosolic calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) induced by DMPP were also inhibited by the extract. However, the binding of [(3)H]nicotine to nAChRs was not affected by the addition of the extract in receptor binding competition analysis, suggesting that active components in the extract and nicotine do not share the binding site in the nAChR. On the other hand, [Ca(2+)](i)increases induced by high K(+), ionomycin, bradykinin, angiotensin II, and thapsigargin were not inhibited by the extract. The data suggest that the extract from D. aromatica specifically inhibits catecholamine secretion by blocking nAChR in a noncompetitive manner.     

Effects of (1R,9S)-beta-hydrastine on intracellular calcium concentration in PC12 cells

(1R,9S)-beta-hydrastine (BHS) decreases the basal intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells.(5) This study examined the effects of (1R,9S)-BHS on [Ca(2+)](i) in PC12 cells. (1R,9S)-BHS at 10-100 microM in combination with a high extracellular K+ level (56 mM) inhibited the release of dopamine in a concentration-dependent manner with an IC(50) value of 66.5 microM. BHS at 100 microM inhibited the sustained increase in [Ca(2+)](i) induced by a high K+ level (56 mM), and had an inhibitory effect on the 2 microM nifedipine-induced blockage of the K+ -stimulated sustained increase in [Ca(2+)](i). In addition, (1R,9S)-BHS at 100 microM prevented the rapid and sustained increase in [Ca(2+)](i) elicited by 20 mM caffeine, but did not have an effect on the increase induced by 1 microM thapsigargin, in the presence of external Ca(2+). These results suggest that the active sites of (1R,9S)-BHS are mainly L-type Ca(2+) channels and caffeine-sensitive Ca(2+)-permeable channels in PC12 cells.     

Effects of nonylphenol on the calcium signal and catecholamine secretion coupled with nicotinic acetylcholine receptors in bovine adrenal chromaffin cells

Nonylphenol (NP) is the most critical metabolite of alkylphenol polyethoxylate detergents. NP is known as an endocrine disruptor with estrogenic activities and as an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Estrogen has modulatory roles on ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs). Ca(2+)-ATPase inhibitors can modulate the cytosolic calcium concentration ([Ca(2+)](c)]) and thus can affect the calcium signaling coupled with nAChRs. Therefore, NP is predicted to have complex effects on the Ca(2+) signaling and secretion coupled with nAChRs. This study investigated these effects using bovine adrenal chromaffin cells. The results show that NP suppressed the Ca(2+) signaling coupled with nAChRs and voltage-operated Ca(2+) channels in a dose-dependent manner, with IC(50)s of 1 and 5.9 microM, respectively. Estradiol exhibits similar suppression but much lower inhibitory potencies. NP alone induced a transient rise in [Ca(2+)](c) in the presence or absence of extracellular calcium. Thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, partially suppressed the [Ca(2+)](c) rise induced by NP, but NP totally blocked the [Ca(2+)](c) rise induced by thapsigargin. This illustrates that NP can cause Ca(2+) release from thapsigargin-insensitive pools. Thapsigargin suppressed the Ca(2+) signaling coupled with nAChRs but increased that coupled with voltage-operated Ca(2+) channels. We propose that three routes are responsible for the effects of NP on nAChRs: named receptor channels, voltage-gated Ca(2+) channels, and Ca(2+)-induced Ca(2+) release. Three routes are related to the characteristics of NP as steroid-like compounds and Ca(2+)-ATPase inhibitor.     

Novel effect of Y-24180, a presumed specific platelet-activating factor receptor antagonist, on Ca2+ levels and growth of human osteosarcoma cells.

In human osteosarcoma MG63 cells, the effect of Y-24180, a presumed specific platelet-activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) and proliferation was measured by using fura-2 and tetrazolium as fluorescent dyes, respectively. Y-24180 (1-5 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was inhibited by 35% by dihydropyridines or removal of extracellular Ca(2+), but was not altered by verapamil and diltiazem. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which 5 microM Y-24180 failed to increase [Ca(2+)](i); conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited histamine-induced, but not 5 microM Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with 0.1-5 microM Y-24180 inhibited cell proliferation in a concentration-dependent manner. Together, these findings suggest that Y-24180 acts as a potent and cytotoxic Ca(2+) mobilizer in human osteosarcoma cells, by inducing both extracellular Ca(2+) influx and intracellular Ca(2+) release. Alterations in cytosolic Ca(2+) regulation may lead to interferences of various cellular functions; thus, attention should be exercised in using Y-24180 as a selective PAF receptor antagonist.