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.     

Choline induces Ca2+ entry in cultured sympathetic neurones isolated from rat superior cervical ganglion

Choline has been shown to be a specific agonist at alpha7 nicotinic acetylcholine receptors, which are the most Ca(2+) permeable of the ionotropic receptor channels. Whole-cell patch recording combined with the measurement of intracellular free Ca(2+) concentration ([Ca(2+)](i), using Indo1, in cultured rat superior cervical ganglion neurones demonstrated that application of choline induced a slowly desensitizing inward current and increased [Ca(2+)](i). The effect was dose dependent with an EC(50) of 1.6 mM and an n(H) of 1.19. The relationship between the elevation of [Ca(2+)](i) (Delta[Ca(2+)](i)) and charge transfer analysed under various recording conditions showed that the Delta[Ca(2+)](i) induced by choline resulted from an influx of Ca(2+) through nicotinic acetylcholine receptors. The effect of choline on the membrane current and Delta[Ca(2+)](i) was not affected by either short application or pretreatment with alpha-bungarotoxin (50 nM) and methyllycaconitine (1 nM), two alpha7 nicotinic receptors antagonists. These results indicate that activation of non-alpha7 nicotinic acetylcholine receptors by choline significantly increases the Ca(2+) concentration in rat superior cervical ganglion neurones.     

Cotinine and nicotine inhibit each other's calcium responses in bovine chromaffin cells

Cotinine is the major metabolite of nicotine. It has some biological activity, but its pathophysiological effects are largely unclear. We studied whether cotinine initiates calcium transients or affects those induced by nicotine. In bovine adrenal chromaffin cells labeled with the fluorescent calcium indicator Fura 2, cotinine (0. 32-3.2 mM) concentration-dependently increased the intracellular Ca(2+) concentration ([Ca(2+)](i)). The effect was abolished by omitting extracellular Ca(2+) during the stimulations. Also nicotinic receptor channel blockers hexamethonium (10 microM-1 mM) and chlorisondamine (100 microM), as well as a competitive nicotinic receptor antagonist dihydro-beta-erythroidine (10-100 microM), inhibited the response. Cotinine (0.32-3.2 mM) preincubation for 2 min inhibited both the nicotine-induced and the cotinine-induced increases in [Ca(2+)](i). Also nicotine (3.2-10 microM) inhibited the cotinine-induced increase in [Ca(2+)](i). Tetrodotoxin (1 microM) and thapsigargin (1 microM) pretreatments did not affect the responses to cotinine, while 300 nM nimodipine partially inhibited the cotinine-induced increase in [Ca(2+)](i). The results indicate that cotinine has nicotine-like effects on chromaffin cells. It may also desensitize the nicotinic cholinergic receptors, possibly by acting as a low-affinity agonist at these receptors.     

Inhibition by pregnenolone sulfate of nicotinic acetylcholine response in adrenal chromaffin cells

To evaluate whether pregnenolone sulfate, an abundant neurosteroid in the brain, modulates nicotinic receptor-mediated responses, the effect of pregnenolone sulfate on acetylcholine-induced catecholamine secretion was investigated in cultured bovine adrenal chromaffin cells. Pregnenolone sulfate inhibited acetylcholine-induced catecholamine secretion (IC(50): 27 microM). In addition, pregnenolone sulfate inhibited acetylcholine-induced Na(+) (IC(50): 12 microM) and Ca(2+) (IC(50): 20 microM) influxes. However, pregnenolone sulfate did not inhibit either catecholamine secretion or Ca(2+) influx stimulated by high K(+). Binding of [3H]nicotine to nicotinic receptors was not altered by pregnenolone sulfate. The inhibitory effect on the acetylcholine-induced secretion was insurmountable by increasing acetylcholine concentrations, but was enhanced by decreasing external Na(+) concentrations. These results suggest strongly that pregnenolone sulfate noncompetitively inhibits nicotinic receptor-operated ion channels, thereby suppressing Na(+) influx through the channels and, consequently, attenuates both Ca(2+) influx and catecholamine secretion. Our results further indicate that pregnenolone sulfate may modulate nicotinic receptor-mediated responses in the brain.     

ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]ET-1 in a biphasic manner, in contrast to an ET(B)-selective agonist, IRL-1620, that was ineffective. The ET(B)-selective antagonist, BQ-788, inhibited [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET(A) and -ET(B) antibodies confirmed a predominant expression of the ET(A) receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca(2+) resulted in a shift to the right of the ET-1 concentration-response curve. Both the L-type voltage-operated Ca(2+) channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET(A) receptors are expressed and functionally coupled to rise of [Ca(2+)](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca(2+)](i) increase is triggered by Ca(2+) release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca(2+) channels and ryanodine receptors participate in sustaining the Ca(2+) response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca(2+)](i) increase.     

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.     

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.     

H1-histaminergic activation of catecholamine release by chromaffin cells

Bovine adrenal medullary chromaffin cells, prelabeled with [3H]norepinephrine, released a large proportion of cellular 3H-labeled catecholamines (CAs) when stimulated with nicotine, K+, histamine, gamma-aminobutyric acid (GABA) and several peptidic hormones [bradykinin, angiotensin II, thyrotropin releasing hormone (TRH) and neurotensin]. The histamine-induced response was dose dependent and occurred through H1 histaminergic receptors. Quantitatively and temporally the histamine- and nicotine-induced responses differed. Nicotine, during the first minutes, induced a large increase of [3H]CAs, but this response was desensitized rapidly. In contrast, histamine initially provoked a smaller release of [3H]CAs than nicotine but, with prolonged exposure (hours), a much greater response was found with histamine. Moreover, little desensitization was observed with histamine even during extended stimulation. External Ca2+ was obligatory for the histamine response, and both inorganic (Co2+ and Ni2+) and organic (verapamil, nifedipine and D-600) Ca2+ channel blockers significantly reduced release of [3H]CAs. These studies suggest that histamine as well as certain other neuroactive substances could play an important role in the physiology and biochemistry of adrenal medullary chromaffin cells.     

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.     

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.     

Anandamide and NADA bi-directionally modulate presynaptic Ca2+ levels and transmitter release in the hippocampus

BACKGROUND AND PURPOSE: Inhibitory CB(1) cannabinoid receptors and excitatory TRPV(1) vanilloid receptors are abundant in the hippocampus. We tested if two known hybrid endocannabinoid/endovanilloid substances, N-arachidonoyl-dopamine (NADA) and anandamide (AEA), presynapticaly increased or decreased intracellular calcium level ([Ca(2+)](i)) and GABA and glutamate release in the hippocampus. EXPERIMENTAL APPROACH: Resting and K(+)-evoked levels of [Ca(2+)](i) and the release of [(3)H]GABA and [(3)H]glutamate were measured in rat hippocampal nerve terminals. KEY RESULTS: NADA and AEA per se triggered a rise of [Ca(2+)](i) and the release of both transmitters in a concentration- and external Ca(2+)-dependent fashion, but independently of TRPV(1), CB(1), CB(2), or dopamine receptors, arachidonate-regulated Ca(2+)-currents, intracellular Ca(2+) stores, and fatty acid metabolism. AEA was recently reported to block TASK-3 potassium channels thereby depolarizing membranes. Common inhibitors of TASK-3, Zn(2+), Ruthenium Red, and low pH mimicked the excitatory effects of AEA and NADA, suggesting that their effects on [Ca(2+)](i) and transmitter levels may be attributable to membrane depolarization upon TASK-3 blockade. The K(+)-evoked Ca(2+) entry and Ca(2+)-dependent transmitter release were inhibited by nanomolar concentrations of the CB(1) receptor agonist WIN55212-2; this action was sensitive to the selective CB(1) receptor antagonist AM251. However, in the low micromolar range, WIN55212-2, NADA and AEA inhibited the K(+)-evoked Ca(2+) entry and transmitter release independently of CB(1) receptors, possibly through direct Ca(2+) channel blockade. CONCLUSIONS AND IMPLICATIONS: We report here for hybrid endocannabinoid/endovanilloid ligands novel dual functions which were qualitatively similar to activation of CB(1) or TRPV(1) receptors, but were mediated through interactions with different targets.     

Effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine release in bovine adrenal chromaffin cells

1 The effects of genistein and herbimycin, tyrosine kinase inhibitors, on catecholamine (CA) release were examined in bovine adrenal chromaffin cells. 2 In intact cells, genistein (10-100 microm) and herbimycin (3-30 microm) inhibited CA release induced by acetylcholine (ACh; 100 microm) or the nicotinic receptor stimulant 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 10 microm), but did not affect CA release induced by high K+ (40 mm). 3 Genistein and herbimycin inhibited (45)Ca2+ uptake induced by ACh (100 microm). 4 Neither genistein nor herbimycin affected [(3)H]nicotine binding with nicotinic receptors. 5 In beta-escin-permeabilized cells, neither genistein nor herbimycin affected CA release induced by Ca2+ (1 microm). 6 These results suggest that protein tyrosine kinase plays the facilitatory role in the regulation of CA release induced by nicotinic receptor stimulation in stimulus-secretion coupling of bovine adrenal chromaffin cells.     

Noncompetitive inhibition by camphor of nicotinic acetylcholine receptors

The effect of camphor, a monoterpenoid, on catecholamine secretion was investigated in bovine adrenal chromaffin cells. Camphor inhibited [3H]norepinephrine ([3H]NE) secretion induced by a nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), with a half-maximal inhibitory concentration (IC50) of 70 +/- 12 microM. In addition, camphor inhibited the rise in cytosolic calcium ([Ca2+]i) and sodium ([Na+]i) induced by DMPP with IC50 values of 88 +/- 32 and 19 +/- 2 microM, respectively, suggesting that the activity of nAChRs is also inhibited by camphor. On the other hand, binding of [3H]nicotine to nAChRs was not affected by camphor. [Ca2+]i increases induced by high K+, veratridine, and bradykinin were not affected by camphor. The data suggest that camphor specifically inhibits catecholamine secretion by blocking nAChRs without affecting agonist binding.     

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.     

N-(4-Trifluoromethylphenyl)amide group of the synthetic histamine receptor agonist inhibits nicotinic acetylcholine receptor-mediated catecholamine secretion

The therapeutic targeting of nicotinic receptors requires the identification of drugs that selectively activate or inhibit a limited range of nicotine acetylcholine receptors (nAChRs). In this study, we identified N-(4-trifluoromethylphenyl)amide group of the synthetic histamine receptor ligands, histamine-trifluoromethyltoluide, that act as potent inhibitors of nAChRs in bovine adrenal chromaffin cells. Catecholamine secretion induced by the nAChRs agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), was significantly inhibited by histamine-trifluoromethyltoluide. Real time carbon-fiber amperometry confirmed the ability of histamine-trifluoromethyltoluide to inhibit DMPP-induced exocytosis in single chromaffin cells. We also found that histamine-trifluoromethyltoluide inhibited DMPP-induced [Ca(2+)](i) and [Na(+)](i) increases, as well as DMPP-induced inward currents in the absence of extracellular calcium. Histamine-trifluoromethyltoluide had no effect on [(3)H]nicotine binding or on calcium increases induced by high K(+), bradykinin, veratridine, histamine, and benzoylbenzoyl ATP. Among the synthetic histamine receptor ligands, clobenpropit exhibited similarity. In addition, 4'-nitroacetanilide also significantly attenuated nAChR-mediated catecholamine secretion. In conclusion, the N-(4-trifluoromethylphenyl)amide group of the histamine-trifluoromethyltoluide might be the critical moiety in the inhibition of nAChR-mediated CA secretion.     

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.     

Effect of celecoxib on Ca2+ movement and cell proliferation in human osteoblasts

In human osteoblasts, the effect of the widely prescribed cyclooxygenase-2 inhibitor celecoxib on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cell proliferation was explored by using fura-2 and the tetrazolium assay, respectively. Celecoxib at concentrations greater than 1microM caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner ( EC 50= 10 microM). Celecoxib-induced [Ca(2+)](i) rise was reduced by 90% by removal of extracellular Ca(2+), and by 30% by l-type Ca(2+) channel blockers. Celecoxib-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that celecoxib-induced extracellular Ca(2+) influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of celecoxib on [Ca(2+)](i) was greatly inhibited. Conversely, pretreatment with celecoxib to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phoispholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not celecoxib-induced, [Ca(2+)](i) rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, partly inhibited celecoxib-induced [Ca(2+)](i) rise in Ca(2+)-containing medium. Separately, overnight treatment with 1-100microM celecoxib inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human osteoblasts, celecoxib increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Celecoxib may be cytotoxic at higher concentrations.     

Specific binding of inositol hexakisphosphate (phytic acid) to adrenal chromaffin cell membranes and effects on calcium-dependent catecholamine release.

In a membrane preparation of cultured bovine adrenal chromaffin cells, [3H]inositol hexakisphosphate ([3H]InsP6) was shown to bind specifically with a Kd of 90 nM and a Bmax of 700 fmol/mg protein. The Hill coefficient was not significantly different from unity. The association of [3H]InsP6 was slow, with equilibrium binding being reached within 10 min. The dissociation of [3H]InsP6 showed monophasic kinetics. In [3H]InsP6 competition binding experiments, we found that reduction in the number of phosphorylated sites in inositol resulted in a gradual loss of binding potency. In intact bovine adrenal chromaffin cells, InsP6 elicited a concentration-dependent facilitation of 45C2+ influx along with the release of the catecholamines, epinephrine and norepinephrine. The latter responses were slower and longer-lasting than responses to depolarizing stimuli, such as nicotine and high K+. The catecholamine release required the presence of extracellular Ca2+. In good agreement with the binding studies, lower inositol phosphates displayed reduced secretagogue potency. In conclusion, in bovine adrenal chromaffin cells, InsP6 appeared to bind to specific sites and elicited Ca2+ influx and catecholamine release.     

2,4-Toluene diisocyanate suppressed the calcium signaling of ligand gated ion channel receptors

Toluene diisocyanate (TDI) is widely used as a chemical intermediate in the production of polyurethane. TDI-induced asthma is related to its disturbance of acetylcholine activity in most affected workers, but the relevant mechanisms are unclear. Toluene diamine (TDA) is the main metabolite of TDI. TDI and TDA have in common the basic toluene structure. Toluene is an abused solvent affecting neuronal signal transduction by influencing the function of ligand gated ion channel receptors, including nicotinic acetylcholine receptors (nAChR), P2X purinoceptors, [gamma]-aminobutyric acid type A (GABAA) receptors, etc. To understand the actions of TDI and TDA on ligand gated ion channels, we investigated their effects on the changes of cytosolic calcium concentration ([Ca2+]c) while stimulating nAChR in human neuroblastoma SH-SY5Y cells, P2 purinoceptors in PC12 cells, and GABAA receptors in bovine adrenal chromaffin cells. Our results showed that both TDI and TDA suppressed the [Ca2+]c rise induced by the potent nicotinic ligand, epibatidine, in human SH-SY5Y cells. Similar but stronger suppression of ATP-induced [Ca2+]c rise occurred in PC12 cells. TDI and TDA also partially suppressed the [Ca2+] c rise induced by GABA in bovine adrenal chromaffin cells. We conclude that TDI and TDA can act on ligand gated ion channel receptors. Our findings suggest that TDI and TDA might have some neurotoxicity that will need to be investigated.     

Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y cells

The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca(2+)](c)) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca(2+) mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca(2+)](c) by releasing Ca(2+) from the intracellular stores and extracellular Ca(2+) influx. 500 microM TDI induced a net [Ca(2+)](c) increase of 112+/-8 and 78+/-6 nM in the presence and absence of extracellular Ca(2+), respectively. In Ca(2+)-free buffer, TDI induced Ca(2+) release from internal stores to reduce their Ca(2+) content and this reduction was evidenced by a suppression occurring on the [Ca(2+)](c) rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca(2+), simultaneous exposure to TDI and methacholine led a higher level of [Ca(2+)](c) compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca(2+)](c) homeostasis including releasing Ca(2+) from internal stores and inducing extracellular Ca(2+) influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.