Effects of capsaicin on Ca(2+) release from the intracellular Ca(2+) stores in the dorsal root ganglion cells of adult rats

We have investigated the effect of capsaicin on Ca(2+) release from the intracellular calcium stores. Intracellular calcium concentration ([Ca(2+)](i)) was measured in rat dorsal root ganglion (DRG) neurons using microfluorimetry with fura-2 indicator. Brief application of capsaicin (1 microM) elevated [Ca(2+)](i) in Ca(2+)-free solution. Capsaicin-induced [Ca(2+)](i) transient in Ca(2+)-free solution was evoked in a dose-dependent manner. Resiniferatoxin, an analogue of capsaicin, also raised [Ca(2+)](i) in Ca(2+)-free solution. Capsazepine, an antagonist of capsaicin receptor, completely blocked the capsaicin-induced [Ca(2+)](i) transient. Caffeine completely abolished capsaicin-induced [Ca(2+)](i) transient. Dantrolene sodium and ruthenium red, antagonists of the ryanodine receptor, blocked the effect of capsaicin on [Ca(2+)](i). However, capsaicin-induced [Ca(2+)](i) transient was not affected by 2-APB, a membrane-permeable IP(3) receptor antagonist. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by bradykinin and phospholipase C inhibitors, neomycin, and U-73122, did not block capsaicin-induced [Ca(2+)](i) transient. In conclusion, capsaicin increases [Ca(2+)](i) through Ca(2+) release from ryanodine-sensitive Ca(2+) stores, but not from IP(3)-sensitive Ca(2+) stores in addition to Ca(2+) entry through capsaicin-activated nonselective cation channel in rat DRG neurons.     

The cytosolic calcium concentration is affected by S-nitrosocysteine in human lymphomonocytes

The homeostasis of cytosolic calcium [Ca2+](c) in mammalian cells is a complex phenomenon, requiring the contribution of many cellular and extracellular systems. Nitric oxide (NO) acts on [Ca2+](c), although the mechanism of this action is unknown. We study the release and the uptake of Ca2+ in the endoplasmic reticulum and its capacitative entry in human lymphomonocytes in the presence of the NO donor S-nitrosocysteine (CysNO) at low (16 microM) and at high (160 microM) concentrations by measuring the [Ca2+](c) by the Fura 2-AM method. Thapsigargin (TG), which inhibits sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and nifedipine (NIF), which blocks the Ca2+ release from intracellular stores, are used to clarify the effects of NO on calcium movements. In the absence of extracellular Ca2+, CysNO decreases basal [Ca2+](c), whereas TG increases it as the result of SERCA inhibition. This effect of TG diminishes in the presence of the NO donor. In the presence of extracellular Ca2+(capacitative entry conditions), CysNO does not influence Ca2+ entry but reduces the toxic effects of TG connected to the increase of [Ca2+](c) in these conditions. The effect of NIF is, up to a certain extent, similar to that of CysNO, although the mechanisms of action of the two agents do not seem related. We conclude that CysNO participates in [Ca2+](c) homeostasis by stimulating the movement of the ion from the cytosol to other compartments.     

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.     

Arachidonic acid regulates two Ca2+ entry pathways via nitric oxide

Several regulated Ca2+ entry pathways have been identified, with capacitative Ca2+ entry (CCE) being the most characterized. In the present study, we examined Ca2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca2+ release from intracellular stores, and increased Ca2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca2+ entry when CCE was blocked by gadolinium (Gd3+). AA-induced Ca2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca2+ release and Ca2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca2+, and to activate a second Ca2+ entry pathway that is dependent on depletion of Ca2+ from ryanodine-sensitive stores.     

Sulfhydryl modification induces calcium entry through IP₃-sensitive store-operated pathway in activation-dependent human neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT₁MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca²⁺]_c) without external calcium, followed by a second [Ca²⁺]_c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca²⁺]_c change of Ca²⁺-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca²⁺]_c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP₃-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.     

Growth inhibition of capsaicin on HeLa cells is not mediated by intracellular calcium mobilization

The effects of capsaicin on cellular growth and intracellular calcium mobilization were examined in human cervical carcinoma derivation, HeLa cells. Capsaicin inhibited cellular growth and increased intracellular calcium level in HeLa cells. This capsaicin-induced intracellular calcium concentration rise was blocked by capsazepin, vanilloid (capsaicin) receptor antagonist. But, an intracellular calcium chelator BAPTA/AM did not block the inhibitory effect of capsaicin on cellular growth. These observations suggest that intracellular calcium mobilization is not required for the capsaicin-induced inhibition of cellular growth.     

The Effect of Spider Toxin PhTx3-4, ω-Conotoxins MVIIA and MVIIC on Glutamate Uptake and on Capsaicin-Induced Glutamate Release and [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in Spinal cord Synaptosomes

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and ω-conotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or ω-conotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca²⁺-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca²⁺]_i in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.     

Evidence for a pertussis toxin sensitive calcium entry pathway in thyroid FRTL-5 cells

Receptor-mediated calcium entry was investigated in Fura 2 loaded FRTL-5 cells. The purinergic agonist ATP activated the release of sequestered calcium and the entry of extracellular calcium. Downregulation of protein kinase C (PKC) substantially enhanced the ATP-evoked calcium entry. Pretreatment of the cells with pertussis toxin (Ptx) decreased the ATP-evoked calcium entry by 56% and the release of sequestered calcium by 34%. In PKC-downregulated cells, the effect of Ptx treatment on the ATP-evoked increase in [Ca²⁺]_iwas 73% and 44%, respectively. Phorbol myristic acetate (PMA) decreased the ATP-evoked calcium entry to the same extent as Ptx. In Ptx-treated cells, the ATP-evoked influx of ⁴⁵Ca²⁺ was attenuated. Stimulation of the cells with P_2p-purinergic agonist GTP evoked no entry of calcium, although GTP released the same amount of sequestered calcium as did ATP. PKC downregulation or pretreatment with Ptx had no effects on the GTP-evoked responses, whereas PMA decreased the GTP-evoked release of calcium. We conclude that the ATP-activated rapid calcium entry pathway is a second messenger-operated calcium channel. © 1995 Wiley-Liss, Inc.     

A nitric oxide/cyclic GMP-dependent protein kinase pathway alters transmitter release and inhibition by somatostatin at a site downstream of calcium entry

We have examined the somatostatin-mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium-dependent manner by a high potassium (55 mM KCI) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCI exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pre-treatment with pharmacologic agents that selectively block cyclic GMP (cGMP)-dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin-mediated acetylcholine release inhibition occurred without reversal of the somatostatin-mediated block of the KCl-evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCI-evoked acetylcholine release, but only cGMP could reduce the KCI-evoked calcium transient. Although cGMP could reduce the KCI-evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore-evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO-PKG pathway can inhibit acetylcholine release, and alter somatostatin-mediated inhibition, by affecting transmitter release at some point after calcium entry.     

Modulatory effect of cyclic AMP on calcium fluxes in FRTL-5 cells

The aim of the present study was to investigate the effect of cAMP on calcium fluxes in Fura 2 loaded thyroid FRTL-5 cells. Preincubating the cells with the phosphodiesterase inhibitor Ro-201724 decreased the ATP-stimulated entry of calcium, while having no effect on the release of sequestered calcium. Pretreatment with forskolin decreased both the release of sequestered calcium and the entry of calcium in response to ATP. We then incubated the cells with phenylisopropyl adenosine (PIA), a P₂₁-receptor agonist earlier shown to decrease cAMP in FRTL-5 cells. Although we did not observe a decrease in cellular cAMP after PIA, the ATP-evoked calcium response was enhanced. Forskolin decreased calcium entry induced by thapsigargin a Ca^2?-ATPase inhibitor, but forskolin had no effect on the thapsigargin-evoked release of sequestered calcium. Addition of calcium to cells stimulated with ATP in a calcium-free buffered resulted in a rapid influx of calcium. This response in [Ca²⁺]_i was decreased in cells pretreated with forskolin. In cells stimulated with thapsigargin, the increase in [Ca²⁺]_i after addition of calcium was inhibited in part by forskolin and enhanced by PIA. The results suggest that cAMP may regulate calcium fluxes in FRTL-5 cells Furthermore, PIA increased agonist-induced calcium entry through a presently unknown mechanism. © 1993 Wiley-Liss, Inc.     

Canonical Transient Receptor Potential Channel 2 (TRPC2) as a Major Regulator of Calcium Homeostasis in Rat Thyroid FRTL-5 Cells: IMPORTANCE OF PROTEIN KINASE C δ (PKCδ) AND STROMAL INTERACTION MOLECULE 2 (STIM2)*

Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCβ1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca²⁺-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca²⁺-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.     

Thapsigargin-induced calcium entry in FRTL-5 cells: Possible dependence on phospholipase A2 activation

Stimulating rat thyroid FRTL-5 cells with agonists that activate the inositol phosphate cascade results in the release of sequestered calcium and influx of extracellular calcium. In addition, phospholipase A₂ (PLA₂) is activated. Since PLA₂ is a calcium-dependent enzyme we wanted to investigate the interrelationships between PLA₂ activity and the entry of calcium. Stimulating ³H-arachidonic acid (³H-AA)-labelled cells with thapsigargin resulted in a substantial release of ³H-AA. This release was totally abolished in a calcium-free buffer. Pretreatment of Fura 2 loaded cells with 4-bromophenacyl bromide, an inhibitor of PLA₂ activity, decreased the thapsigargin-induced entry of calcium, suggesting a role for PLA₂ in the regulation of calcium entry. In cells treated with nordihydroguaiaretic acid (NDGA), clotramizole, or econazole, compounds with lipoxygenase and cytochrome P-450 inhibitory actions, the thapsigargin-induced entry of calcium was decreased in a dose-dependent manner. However, treatment of the cells with indomethacin, a cyclooxygenase inhibitor, had no effect on the thapsigargin-induced calcium entry. We also showed that stimulation of the cells with arachidonic acid released sequestered calcium, apparently from the same intracellular pool as did thapsigargin. The results suggested that the calcium-induced PLA₂ activation and the metabolism of the produced arachidonic acid by a noncyclooxygenase pathway may be of importance in maintaining calcium entry after releasing sequestered Ca²⁺ in FRTL-5 cells. © 1994 Wiley-Liss, Inc.     

Growth-dependent modulation of capacitative calcium entry in normal rat kidney fibroblasts

Normal rat kidney (NRK) fibroblasts have electrophysiological properties and intracellular calcium dynamics that are dependent upon their growth stage. In the present study we show that this differential behavior coincides with a differential calcium entry that can be either capacitative or non-capacitative. Confluent cells made quiescent by serum deprivation, which have a stable membrane potential near ? 70 mV and do not show spontaneous intracellular calcium oscillations, primarily exhibit the capacitative mechanism for calcium entry, also called store-operated calcium entry (SOCE). When the quiescent cells are grown to density-arrest in the presence of EGF as the sole polypeptide growth factor, these cells characteristically fire spontaneously repetitive calcium action potentials, which propagate throughout the whole monolayer and are accompanied by intracellular calcium transients. These density-arrested cells appear to exhibit in addition to SOCE also receptor-operated calcium entry (ROCE) as a mechanism for calcium entry. Furthermore we show that, in contrast to earlier studies, the employed SOCs and ROCs are permeable for both calcium and strontium ions. We examined the expression of the canonical transient receptor potential channels (Trpcs) that may be involved in SOCE and ROCE. We show that NRK fibroblasts express the genes encoding Trpc1, Trpc5 and Trpc6, and that the levels of their expression are dependent upon the growth stage of the cells. In addition we examined the growth stage dependent expression of the genes encoding Orai1 and Stim1, two proteins that have recently been shown to be involved in SOCE. Our results suggest that the differential expression of Trpc5, Trpc6, Orai1 and Stim1 in quiescent and density-arrested NRK fibroblasts is responsible for the difference in regulation of calcium entry between these cells. Finally, we show that inhibition or potentiation of SOCE and ROCE by pharmacological agents has profound effects on calcium dynamics in NRK fibroblasts.     

Estrogen and the Ca2+-mobilizing agonist ATP evoke acute NO synthesis via distinct pathways in an individual human vascular endothelium-derived cell

In this study, we have systematically evaluated the signaling mechanisms underlying stimulated nitric oxide (NO) synthesis by estrogen (E2) and other vasoactive agents at the level of a single endothelium-derived cell. To do so, we have characterized and contrasted rapid E2-evoked NO synthesis with that of ATP using single-cell microfluorimetry and patch-clamp recordings to monitor stimulated changes in cellular NO synthesis (via 4-amino-5-methylamino-2',7'-difluorofluorescein), Ca2+ transients (via Fluo-3), and membrane hyperpolarization in cultured human EA.hy926 cells. E2-evoked NO synthesis in single cells (EC50 approximately 0.3 nM) was blocked by the E2 receptor antagonist ICI 182,780 and the NO synthase inhibitor N(omega)-nitro-l-arginine methyl ester. Although both E2 and ATP stimulated comparable Ca2+ transients, E2-induced NO synthesis was insensitive to intracellular BAPTA-AM or removal of external Ca2+. In contrast, ATP-evoked NO production was abolished by either one of these treatments. ATP-evoked hyperpolarizations ( approximately 20 mV) and NO production were both inhibited by the respective small-conductance and intermediate-conductance calcium- activated K+ channel blockers apamin and charybdotoxin. E2 minimally affected membrane potential, and stimulated NO synthesis was insensitive to calcium-activated K+ channel blockers. Exposure to either the phosphatidylinositol 3-kinase inhibitor LY-294002 or the MAP kinase inhibitor PD-98059 abolished the NO response to E2, but not that to ATP. Finally, the NO response evoked by a combined stimulus of E2 plus ATP was similar to that of ATP alone. In conclusion, our data directly demonstrate that an individual human EA.hy926 cell contains at least two distinct mechanisms for stimulated NO synthesis that depend on either calcium or protein kinase signaling events.     

Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2+ entry, optimal influx of Ca2+, and degranulation in a rat mast cell line

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca(2+) and the ensuing influx of external Ca(2+), also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca(2+) because Sr(2+) and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca(2+) and Sr(2+) as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca(2+) but not Sr(2+), whereas overexpression of STIM1 and TRPC5 allows entry of both Ca(2+) and Sr(2+). These and other observations suggest that the Sr(2+)-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca(2+) to generate a signal for degranulation.     

Moderate extracellular acidification inhibits capsaicin-induced cell death through regulating calcium mobilization, NF-κB translocation and ROS production in synoviocytes

We previously show the expression of transient receptor potential vanilloid 1 (TRPV1) in primary synoviocytes from collagen-induced arthritis (CIA) rats. Capsaicin and lowered extracellular pH from 7.4 to 5.5 induce cell death through TRPV1-mediated Ca(2+) entry and reactive oxygen species (ROS) production. However, under the pathological condition in rheumatoid arthritis, the synovial fluid is acidified to a moderate level (about pH 6.8). In the present study, we examined the effects of pH 6.8 on the TRPV1-mediated cell death. Our finding is different or even opposite from what was observed at pH 5.5. We found that the moderate extracellular acidification (from pH 7.4 to 6.8) inhibited the capsaicin-induced Ca(2+) entry through attenuating the activity of TRPV1. In the mean time, it triggered a phospholipse C (PLC)-related Ca(2+) release from intracellular stores. The nuclear translocation of NF-κB was found at pH 6.8, and this also depends on PLC activation. Moreover, the capsaicin-evoked massive ROS production and cell death were depressed at pH 6.8, both of which are dependent on the activation of PLC and NF-κB. Taken together, these results suggested that the moderate extracellular acidification inhibited the capsaicin-induced synoviocyte death through regulating Ca(2+) mobilization, activating NF-κB nuclear translocation and depressing ROS production.     

Phosphatase inhibition reveals a calcium entry pathway dependent on protein kinase A in thyroid FRTL-5 cells: comparison with store-operated calcium entry

Calcium entry through store-operated calcium channels is an important entry mechanism. In the present report we have described a novel calcium entry pathway that is independent of depletion of intracellular calcium stores. Treatment of the cells with the phosphatase inhibitor calyculin A (caly A), which blocked thapsigargin-evoked store-operated calcium entry (SOCE), induced a potent concentration-dependent calcium entry. In a calcium-free buffer, acute addition of caly A evoked a very modest increase in cytosolic free calcium ([Ca(2+)](i)). This increase was not from the agonist-mobilizable calcium stores, as the thapsigargin-evoked increase in [Ca(2+)](i) was unaltered in caly A-treated cells. The caly A-evoked calcium entry was not blocked by Gd(3+) or 2-APB, whereas SOCE was. Caly A enhanced the entry of barium, indicating that the increase in intracellular calcium was not the result of a decreased extrusion of calcium from the cytosol. Jasplakinolide and cytochalasin D had only marginal effects on calcium entry. The protein kinase A (PKA) inhibitor H-89 and an inhibitory peptide for PKA abolished the caly A-evoked entry of both calcium and barium. The SOCE was, however, enhanced in cells treated with H-89. In cells grown in the absence of thyrotropin (TSH), the caly A-evoked entry of calcium was smaller compared with cells grown in TSH-containing buffer. Stimulation of cells grown without TSH with forskolin or TSH restored the calyculin A-evoked calcium entry to that seen in cells grown in TSH-containing buffer. SOCE was decreased in these cells. Our results thus suggest that TSH, through the production of cAMP and activation of PKA, regulates a calcium entry pathway in thyroid cells. The pathway is distinctly different from the SOCE. As TSH is the main regulator of thyroid cells, we suggest that the novel calcium entry pathway participates in the regulation of basal calcium levels in thyroid cells.     

Nitrergic relaxation in rat gastric fundus: influence of mechanism of induced tone and possible role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase

The aim of this study was to investigate the influence of the mechanism of induced tone and the role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) in nitrergic relaxation of rat gastric fundus. Prostaglandin F(2alpha) (PGF(2alpha)), thapsigargin (TSG) and cyclopiazonic acid (CPA) were used in concentrations that induced a similar contraction (20 g force/g tissue). Nifedipine (3 x 10(-7) M) completely relaxed PGF(2alpha)-contracted tissues and relaxed tissues contracted by TSG and CPA by 20 +/- 6% and 56 +/- 12% respectively; contraction induced by the three contractile agents was fully reversed by a general Ca2+ entry blocker 1-[2-(4-methoxyphenyl)-2-[3-(4-metoxyphenyl)propoxy]ethyl-1H-imidazole HCl (SKF 96365; 10(-5) M). In the presence of nifedipine (3 x 10(-7) M) or verapamil (10(-5) M), PGF(2alpha) and CPA-induced contractions were still approximately 50% relaxed by SKF 96365. This suggests that contractions induced by PGF(2alpha) are related to Ca2+ entry through L-type voltage-operated Ca2+ channels and that contractions by TSG are mainly related to Ca2+ entry through store-operated Ca2+ channels. Relaxant responses to exogenous nitric oxide (NO), to endogenous NO released by electrical field stimulation, and to vasoactive intestinal polypeptide (VIP) were studied in tissues contracted by TSG and CPA and compared to responses in tissues contracted by PGF(2alpha). Responses to exogenous and endogenous NO were greatly attenuated in TSG-contracted tissues, but not in CPA-contracted tissues. When contraction was induced by CPA in the presence of nifedipine or verapamil, relaxations to exogenous and endogenous NO were also significantly reduced. Relaxation induced by VIP was reduced in tissues contracted by either TSG or CPA in the presence of nifedipine or verapamil. These results suggest that the ability of the nitrergic neurotransmitter to induce relaxation of rat gastric fundus is influenced by the mechanism used to induce tone and are indicative for a role for SERCA in nitrergic relaxation. However, activation of SERCA appears to not be unique for nitrergic relaxation, but might also be used by VIP, a co-transmitter of NO in this tissue.     

Ca(2+) entry activated by S-nitrosylation. Relationship to store-operated ca(2+) entry

The coupling between Ca(2+) pools and store-operated Ca(2+) entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca(2+) entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca(2+) pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T. , and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT(1)MF-2 smooth muscle cells and DC-3F fibroblasts, Ca(2+) entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca(2+) removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca(2+) entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca(2+) entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba(2+) entry and sensitivity to blockade by La(3+) were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca(2+) entry.     

Stimulation of nitric oxide synthase in cerebral cortex due to elevated partial pressures of oxygen: an oxidative stress response

The purpose of this investigation was to determine the impact of elevated partial pressures of O(2) on the steady state concentration of nitric oxide ((*)NO) in the cerebral cortex. Rodents with implanted O(2)- and (*)NO-specific microelectrodes were exposed to O(2) at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA) for up to 45 min. Elevations in (*)NO concentration occurred with all partial pressures above that of ambient air. In rats exposed to 2.8 ATA O(2) the increase was 692 +/- 73 nM (S.E., n = 5) over control. Changes were not associated with alterations in concentrations of nitric oxide synthase (NOS) enzymes. Based on studies with knock-out mice lacking genes for neuronal NOS (nNOS) or endothelial NOS (eNOS), nNOS activity contributed over 90% to total (*)NO elevation due to hyperoxia. Immunoprecipitation studies indicated that hyperoxia doubles the amount of nNOS associated with the molecular chaperone, heat shock protein 90 (Hsp90). Both (*)NO elevations and the association between nNOS and Hsp90 were inhibited in rats infused with superoxide dismutase. Elevations of (*)NO were also inhibited by treatment with the relatively specific nNOS inhibitor, 7 nitroindazole, by the ansamycin antibiotics herbimycin and geldanamycin, by the antioxidant N-acetylcysteine, by the calcium channel blocker nimodipine, and by the N-methyl-D-aspartate inhibitor, MK 801. Hyperoxia did not alter eNOS association with Hsp90, nor did it modify nNOS or eNOS associations with calmodulin, the magnitude of eNOS tyrosine phosphorylation, or nNOS phosphorylation via calmodulin kinase. Cerebral cortex blood flow, measured by laser Doppler flow probe, increased during hyperoxia and may be causally related to elevations of steady state (*)NO concentration. We conclude that hyperoxia causes an increase in (*)NO synthesis as part of a response to oxidative stress. Mechanisms for nNOS activation include augmentation in the association with Hsp90 and intracellular entry of calcium.