Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.     

Characterization using FLIPR of rat vanilloid receptor (rVR1) pharmacology

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.     

Pharmacology of vanilloids at recombinant and endogenous rat vanilloid receptors

This study compared the actions of members of five different chemical classes of vanilloid agonists at the recombinant rat vanilloid VR1 receptor expressed in HEK293 cells, and at endogenous vanilloid receptors on dorsal root ganglion cells and sensory nerves in the rat isolated mesenteric arterial bed. In mesenteric beds, vanilloids elicited dose-dependent vasorelaxation with the rank order of potency: resiniferatoxin>capsaicin=olvanil>phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV)>isovelleral. Scutigeral was inactive. Responses were abolished by capsaicin pretreatment and inhibited by ruthenium red. In VR1-HEK293 cells and dorsal root ganglion neurones, Ca(2+) responses were induced by resiniferatoxin>capsaicin=olvanil>PPAHV; all four were full agonists. Isovelleral and scutigeral were inactive. The resiniferatoxin-induced Ca(2+) response had a distinct kinetic profile. Olvanil had a Hill coefficient of approximately 1 whilst capsaicin, resiniferatoxin and PPAHV had Hill coefficients of approximately 2 in VR1-HEK293 cells. The capsaicin-induced Ca(2+) response was inhibited in a concentration-dependent manner by ruthenium red>capsazepine>isovelleral. These data show that resiniferatoxin, capsaicin, olvanil and PPAHV, but not scutigeral and isovelleral, are agonists at recombinant rat VR1 receptors and endogenous vanilloid receptors on dorsal root ganglion neurones and in the rat mesenteric arterial bed. The vanilloids display the same relative potencies (resiniferatoxin>capsaicin=olvanil>PPAHV) in all of the bioassays.     

The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1)

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.     

Interactions of fluoroquinolone antibacterials, DX-619 and levofloxacin, with creatinine transport by renal organic cation transporter hOCT2

Interactions of DX-619, a novel fluoroquinolone antibacterial, and levofloxacin (LVFX) with the human renal organic cation transporter hOCT2 were studied. The intracellular accumulation of [(14)C]creatinine in stable transfectants of HEK293 cells expressing hOCT2 (hOCT2-HEK293) as well as vector-transfected HEK293 cells (VEC-HEK293) was evaluated in the presence of DX-619 and LVFX at various concentrations. When added extracellularly, both DX-619 and LVFX inhibited the uptake of [(14)C]creatinine (5 microM) by hOCT2-HEK293 cells in a dose-dependent manner. Unlike in hOCT2-HEK293 cells, the uptake in VEC-HEK293 cells was not inhibited by either fluoroquinolone suggesting that hOCT2 was specifically involved in the inhibition. The apparent IC(50) value for the inhibition of [(14)C]creatinine uptake in hOCT2-HEK293 cells was 1.29+/-0.23 microM for DX-619 and 127+/-27 microM for LVFX, indicating DX-619 to be approximately 100-fold more potent than LVFX at inhibiting the transport of [(14)C]creatinine by hOCT2. A Dixon plot revealed that the inhibition by DX-619 of the hOCT2-mediated transport of [(14)C]creatinine was competitive. Fluoroquinolone antibacterials have the ability to inhibit the transport of creatinine by hOCT2, with DX-619 being much more effective than LVFX.     

酪氨酸激酶抑制剂对hTrp3蛋白介导的α_(1B)-AR引起的Ca~(2+)内流的影响

目的　探讨Trp3(transientreceptorpotential 3)蛋白是否参与α1B AR引起的Ca2 + 内流以及酪氨酸激酶对其调控作用。方法　采用脂质体转染 ,将hTrp3cDNA分别转染到HEK2 93细胞和已有α1B受体稳定表达的HEK2 93细胞 ;Westernblot方法检测Trp3蛋白表达情况 ;Fura 2 /AM荧光分光光度法 ,测定胞浆游离Ca2 + 浓度。结果　HEK2 93细胞上可检测到hTrp3的内源性表达 ,转染后其表达增加。α1B HEK2 93细胞转染hTrp3cDNA后 ,α1B AR引起的Ca2 +内流显著增加 (P <0 0 1) ;转染hTrp3cDNA对thapsigargin诱导的Ca2 + 内流无作用。 5～ 30 μmol·L-1genistein对转染细胞α1B AR诱发的Ca2 + 内流有抑制作用 ,最大抑制率达(75 2± 12 6 ) %。结论　Trp3cDNA转染可能主要通过非CRAC(calciumreleaseactivatedcalcium )途径增加α1B AR引起的Ca2 + 内流 ,这一过程很大程度上依赖酪氨酸激酶的调控     

Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay

1. TRPM8 (CMR1) is a Ca(2+)-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3. The EC(50) values of the agonists defined their relative potencies: icilin (0.2+/-0.1 microM)>FrescolatML (3.3+/-1.5 microM) > WS-3 (3.7+/-1.7 microM) >(-)menthol (4.1+/-1.3 microM) >frescolatMAG (4.8+/-1.1 microM) > cooling agent 10 (6+/-2.2 microM) >(+)menthol (14.4+/-1.3 microM) > PMD38 (31+/-1.1 microM) > WS-23 (44+/-7.3 microM) > Coolact P (66+/-20 microM) > geraniol (5.9+/-1.6 mM) > linalool (6.7+/-2.0 mM) > eucalyptol (7.7+/-2.0 mM) > hydroxycitronellal (19.6+/-2.2 mM). 4. Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC(50): 0.8+/-1.0, 3.5+/-1.1 and 18+/-1.1 microM, respectively). 5. The Ca(2+) response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. 6. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.     

Endothelin-induced myoplasmic Ca2+ responses and tyrosine phosphorylation in coronary smooth muscle

This study investigated the role of tyrosine phosphorylation and source of Ca2+ in prolonged endothelin-1 (ET-1)-induced potentiation of myoplasmic free Ca2+ ([Ca2+]m) responses to depolarization in coronary smooth muscle cells. Fura-2 microfluorometry showed typical increases in [Ca2+]m in response to 80 mM K+ (80K) and 0.01 microM endothelin. After washout of ET-1 80K-induced [Ca2+]m increases were augmented (potentiated) 31%. Time to peak [Ca2+]m response to 80K was less after ET-1 exposure than before. ET-1 potentiation of 80K-induced [Ca2+]m responses by decreased sarcoplasmic reticulum (SR) buffering of [Ca2+]m or Ca2+-induced Ca2+ release was ruled out by lack of potentiation by 5 mM caffeine and 1 microM thapsigargin. Diltiazem abolished potentiation, providing evidence for Ca2+ influx through voltage-gated Ca2+ channels (VGCC). Genistein (30 microM) and methyl 2,5-dihydroxycinnamate (1 microM, MDHC) abolished potentiation of Ca2+ influx. Single cell phosphotyrosine measured directly by immunofluorescence was increased 95% in cells treated with ET-1 compared to control, genistein, and MDHC treated cells. ET-1 increased tyrosine phosphorylation of an 80-85 kDa protein, but not the 240 kDa alpha1C subunit of the VGCC. Tyrosine phosphorylation of proteins other than VGCC is necessary for prolonged potentiation by ET-1 of depolarization-induced Ca2+ influx.     

Characterisation using FLIPR of human vanilloid VR1 receptor pharmacology

A full pharmacological characterisation of the recently cloned human vanilloid VR1 receptor was undertaken. In whole-cell patch clamp studies, capsaicin (10 microM) elicited a slowly activating/deactivating inward current in human embryonic kidney (HEK293) cells stably expressing human vanilloid VR1 receptor, which exhibited pronounced outward rectification (reversal potential -2.1+/-0.2 mV) and was abolished by capsazepine (10 microM). In FLIPR-based Ca(2+) imaging studies the rank order of potency was resiniferatoxin>olvanil>capsaicin>anandamide, and all were full agonists. Isovelleral and scutigeral were inactive (1 nM-30 microM). The potencies of capsaicin, olvanil and resiniferatoxin, but not anandamide, were enhanced 2- to 7-fold at pH 6.4. Capsazepine, isovelleral and ruthenium red inhibited the capsaicin (100 nM)-induced Ca(2+) response (pK(B)=6.58+/-0.02, 5.33+/-0.03 and 7.64+/-0.03, respectively). In conclusion, the recombinant human vanilloid VR1 receptor stably expressed in HEK293 cells acted as a ligand-gated, Ca(2+)-permeable channel with similar agonist and antagonist pharmacology to rat vanilloid VR1 receptor, although there were some subtle differences.     

Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide

1. The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [(3)H]-anandamide ([(3)H]-AEA) has been investigated. 2. Palmitoylethanolamide and homologues with chain lengths from 12 - 18 carbon atoms inhibited rat brain [(3)H]-AEA metabolism with pI(50) values of approximately 5. Homologues with chain lengths < or = eight carbon atoms gave < 20% inhibition at 100 microM. 3. R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [(3)H]-AEA metabolism with pI(50) values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively. 4. With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [(3)H]-WIN 55,212-2 binding to human CB(2) receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [(3)H]-CP 55,940 binding to human CB(1) receptors expressed on CHO cells. 5. Most of the compounds had little effect upon the uptake of [(3)H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 microM) and palmitoylisopropylamide (30 and 100 microM) produced more inhibition of [(3)H]-AEA uptake than expected to result from inhibition of [(3)H]-AEA metabolism alone. 6. In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [(3)H]-ethanolamine from [(3)H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective. 7. These data provide useful information upon the ability of palmitoylethanolamide analogues to act as 'entourage' compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB(1) or CB(2) receptors.     

Dual excitatory and inhibitory effects of opioids on intracellular calcium in neuroblastoma x glioma hybrid NG108-15 cells.

The intracellular free calcium concentration ([Ca2+]i) was measured in single NG108-15 cells using indo-1-based microfluorimetry. In cells differentiated for 6-14 days in serum-free, forskolin (5 microM)-supplemented medium, application of micromolar concentrations of [D-Ala2,D-Leu5]-enkephalin (DADLE) inhibited Ca2+ influx mediated by voltage-gated Ca2+ channels. DADLE, at concentrations ranging from 1 nM to 1 microM, also produced rapid transient increases in [Ca2+]i (EC50 = 10 nM). The [Ca2+]i increases elicited by DADLE did not correlate with the inhibitory effects of the peptide. DADLE-induced [Ca2+]i increases were blocked by naloxone. In single cells, sequential application of selective opioid agonists (30 nM) evoked responses of the rank order DADLE = [D-Pen2,D-Pen5]-enkephalin > (trans)-(+-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) benzeneacetamide > [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin, consistent with activation of a delta-opioid receptor. The response was completely blocked by removal of extracellular Ca2+ or application of 1 microM nitrendipine, indicating that the increase in [Ca2+]i results from Ca2+ influx via dihydropyridine-sensitive, voltage-gated Ca2+ channels. Substitution of N-methyl-D-glucamine for extracellular Na+ or application of 1 microM tetrodotoxin greatly reduced, and in some cases blocked, the DADLE-induced [Ca2+]i increase, consistent with amplification of the response by voltage-gated Na+ channels. The [Ca2+]i increase was mimicked by both dibutyryl-cAMP and phorbol 12,13-dibutyrate. These findings are consistent with a delta-opioid-induced depolarization, possibly mediated by a second messenger, that subsequently recruits voltage-sensitive Ca2+ channels. In contrast to differentiated cells, undifferentiated cells responded to DADLE with a modest [Ca2+]i increase that was not sensitive to nitrendipine. In these cells, activation of the same second messenger system may elevate [Ca2+]i by mobilization from intracellular stores rather than influx. In addition to previously described inhibitory coupling to adenylyl cyclase and Ca2+ channels in NG108-15 cells, these results suggest that a novel, excitatory, effector system may also couple to opioid receptors.     

Characterization of interactions of methylmercury with Ca2+ channels in synaptosomes and pheochromocytoma cells: radiotracer flux and binding studies

The interaction of methylmercury (MeHg) with neuronal Ca2+ channels in rat forebrain synaptosomes and dihydropyridine (DHP)-sensitive Ca2+ channels in rat pheochromocytoma (PC12) cells was examined using radiotracer flux assays and radioligand binding analyses. In synaptosomes, the influx of 45Ca2+ was used to examine the voltage and state dependence of block of Ca2+ channels by MeHg, as well as the effects of MeHg on apparent inactivation of 45Ca2+ influx. In addition, the differential influx of 45Ca2+, 85Sr2+, and 133Ba2+ was used to examine the effect of MeHg on the ionic selectivity of synaptosomal Ca2+ channels. The ability of MeHg to block 45Ca2+ influx via a DHP-sensitive Ca2+ channel was examined in PC12 cells. Effects of MeHg on binding of [3H]nitrendipine in synaptosomes and 125I-omega-conotoxin GVIA (CgTx) in synaptosomes and PC12 cells were measured. In synaptosomes, MeHg blocked 45Ca2+ influx in a voltage-dependent manner, inasmuch as increasing the extracellular K+ concentration increased the magnitude of block by 100 microM MeHg. When synaptosomes were incubated for 10 sec in either a nondepolarizing or a depolarizing solution before measurement of 1 sec of depolarization-induced 45Ca2+ influx, the potency and efficacy of the block of 45Ca2+ influx by MeHg were similar. Thus, block of Ca2+ channels by MeHg does not appear to be state dependent. To determine the kinetics of apparent inactivation of 45Ca2+ influx, synaptosomes were predepolarized in Ca2(+)-free high [K+] solution, for intervals varying from 1 to 10 sec, before measurement of 1 sec of K(+)-induced 45Ca2+ influx. When compared with control, MeHg (100 microM) altered the rate constant for apparent inactivation and decreased the fraction of 45Ca2+ influx that does not inactivate. Influx of 45Ca2+, 85Sr2+, and 133Ba2+ during 1 sec of depolarization was blocked in a dose-dependent manner by MeHg, with estimated IC50 values of 125, 150, and greater than 150 microM for 45Ca2+, 85Sr2+, and 133Ba2+, respectively. In triple-label experiments, the relative flux of radiolabeled Ca2+:Sr2+:Ba2+ was altered from approximately 6:2:3 to 6:1:3 in the presence of 100 microM MeHg. In undifferentiated and nerve growth factor-differentiated PC12 cells, K(+)-induced 45Ca2+ influx was blocked by the DHP nifedipine, with an approximate IC50 value of 5 nM. MeHg reduced 45Ca2+ influx in PC12 cells with an estimated IC50 value of 50 microM, and 125 microM MeHg reduced uptake by greater than 90%. [3H]Nitrendipine bound to synaptosomes with high affinity in normal and elevated [K+] solutions.(ABSTRACT TRUNCATED AT 400 WORDS)     

酪氨酸蛋白激酶在不同α_1肾上腺素受体亚型Ca~(2+)调控中的作用

目的　了解酪氨酸蛋白激酶信号途径在不同α1肾上腺素受体亚型Ca2 +调控中的作用。方法　用Fura 2荧光探针双波长测定细胞胞浆游Ca2 +浓度 ([Ca2 +]i)的方法 ,在分别转染了α1A、α1B和α1D肾上腺素受体cDNA的HEK2 93细胞 ,观察酪氨酸蛋白激酶抑制剂Genistein和磷脂酶C抑制剂U7312 2 对激动不同α1肾上腺素受体亚型引起的 [Ca2 +]i 变化的影响。结果　预先用U7312 2 (0 1,10 ,5 0 μmol·L-1)与细胞共同孵育 10min ;用Genistein(10 ,10 0 ,2 0 0 μmol·L-1)与细胞共同孵育 1h。在分别转染了α1A、α1B和α1DcDNA的HEK2 93细胞 ,U73 12 2 和Genistein均能浓度依赖性地抑制肾上腺 (10 μmol·L-1)引起的双相 [Ca2 +]i 的升高。在上述细胞 ,U7312 2 (5 0 μmol·L-1)能完全抑制肾上腺素引起的[Ca2 +]i 升高 ;而用最大有效浓度 10 0 μmol·L-1的Genistein只能部分抑制肾上腺素升高 [Ca2 +]i 的作用。结论　在HEK 2 93细胞 ,不同α1肾上腺素受体亚型 (α1A α1B和α1D)激动均能部分通过酪氨酸蛋白激酶信号途径引起Ca2 +释放和Ca2 +内流。α1肾上腺素受体可能通过G蛋白和酪氨酸蛋白激酶两种途径激活磷脂酶C     

Modulation of sensory neuron potassium conductances by anandamide indicates roles for metabolites

BACKGROUND AND PURPOSE: The endogenous cannabinoid anandamide (AEA) acts at cannabinoid (CB(1)) and vanilloid (TRPV(1)) receptors. AEA also shows antinociceptive properties; although the underlying mechanism for this is not fully understood, both CB(1) and TRPV(1) may be involved. Voltage-activated Ca(2+) channels in rat-cultured dorsal root ganglion (DRG) neurons are modulated by AEA. However, AEA in different populations of neurons enhanced or attenuated KCl-evoked Ca(2+) influx; these effects were linked with soma size. The aim of this study was to determine how AEA or its metabolites might produce these variable responses. EXPERIMENTAL APPROACH: The whole cell patch-clamp technique and fura-2 Ca(2+) imaging were used to characterize the actions of AEA on action potential firing and voltage-activated K(+) currents and to determine whether AEA metabolism plays any role in its effects on cultured DRG neurons. KEY RESULTS: AEA attenuated multiple action potential firing evoked by 300 ms depolarizing current commands in a subpopulation of DRG neurons. Application of 1 microM AEA attenuated voltage-activated K(+) currents and the recovery of KCl-evoked Ca(2+) transients. The insensitivity of these responses to the CB(1) receptor antagonist rimonabant (100 nM) and preincubation of DRG neurons with pertussis toxin suggested that these actions are not CB(1) receptor-mediated. Preincubating DRG neurons with the fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulphonyl fluoride (PMSF) attenuated the inhibitory actions of AEA on K(+) currents and Ca(2+) influx. CONCLUSION AND IMPLICATIONS: These data suggest that the products of AEA metabolism by FAAH contribute to the attenuation of K(+) conductances and altered excitability of cultured sensory neurons.     

The action of pentobarbitone on stimulus-secretion coupling in adrenal chromaffin cells.

The action of pentobarbitone on stimulus-secretion coupling was studied in bovine isolated adrenal medullary cells. Pentobarbitone inhibited catecholamine release evoked by 500 microM carbachol with half maximal inhibition (IC50) around 50 microM. It also inhibited catecholamine release induced by depolarization with 77 mM potassium (IC50 100 microM). These effects of pentobarbitone were observed with concentrations that lie within the range encountered during general anaesthesia. Evoked secretion required the presence of calcium in the extracellular medium and was associated with an influx of Ca2+ through voltage-sensitive channels. Pentobarbitone inhibited 45Ca influx in response to both carbachol (IC50 50 microM) and K+-depolarization (IC50 150 microM). The action of pentobarbitone on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. Catecholamine secretion was measured in the presence of 0, 50, 200 or 500 microM pentobarbitone. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. When catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding pentobarbitone to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. Pentobarbitone inhibited the secretion and 45Ca uptake induced by carbachol in a non-competitive manner. The secretion evoked by nicotinic agonists was associated with an increase in 22Na influx. Pentobarbitone inhibited this influx with an IC50 of 100 microM. We concluded that: (a) Pentobarbitone inhibits the catecholamine secretion from bovine adrenal chromaffin cells induced by nicotinic agonists by non-competitive inhibition of the nicotinic receptor. (b) The decrease in Ca influx caused by pentobarbitone accounts fully for the decrease in secretion in response to depolarization with potassium.     

Requirement of PSD-95 for dopamine D1 receptor modulating glutamate NR1a/NR2B receptor function

Aim： To elucidate the role of scaffold protein postsynaptic density （PSD）-95 in the dopamine D1 receptor （D~R）-modulated NRla/NR2B receptor response. Methods： The human embryonic kidney 293 ceils expressing D1R （tagged with the enhanced yellow fluorescent protein） and NRla/NR2B with or without co-expres- sion of PSD-95 were used in the experiments. The Ca^2＋ influx measured by imaging technique was employed to monitor N-methyl-D-aspartic acid receptors （NMDAR） function. Results： The application of dopamine （DA, 100 μmol/L） did not alter glutamate/glycine （Glu/Gly）-induced NMDAR-mediated Ca^2＋ influx in cells only expressing the D1R/NRla/NR2B receptor. However, DA increased Glu/Glyinduced Ca^2＋ influx in a concentration-dependent manner while the cells were co-expressed with PSD-95. D1R-stimulated Ca^2＋ influx was inhibited by a selective D1R antagonist SCH23390. Moreover, pre-incubation with either the protein kinase A （PKA） inhibitor H89, or the protein kinase C （PKC） inhibitor chelerythrine attenuated D1R-enhanced Ca^2＋ influx induced by the N-methyl-D-aspartic acid （NMDA） agonist. The results clearly indicate that D1R-modulated NRla/NR2B receptor function depends on PSD-95 and is subjected to the regulation of PKA and PKC. Conclusion： The present study provides the first evidence that PSD-95 is essential in D1R-regulated NRla/NR2B receptor function.     

Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide

The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro-2a neuroblastoma and, for comparison, in rat RBL-2H3 basophilic leukaemia cells. Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K(M) values of 28 microM (PEA) and 10 microM (AEA) in Neuro-2a cells, and 30 microM (PEA) and 9.3 microM (AEA) in RBL-2H3 cells. Both PEA and AEA uptake showed temperature-dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride. The AEA uptake was inhibited by AM404, 2-arachidonoylglycerol (2-AG), R1- and S1-methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 microM, did not affect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, (9)-THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA. From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.     

Protein kinase C potentiates capacitative Ca2+ entry that links to steroidogenesis in bovine adrenocortical cells

I investigated the role of protein kinase C (PKC) in regulation of the capacitative Ca2+ entry and steroidogenesis in bovine adrenocortical (BA) cells. Thapsigargin (TG)-treatment depleted intracellular Ca2+ stores followed by induction of Ca2+ influx from the extracellular pool and also increasing of Mn2+ influx as an indicator of divalent cation influx in BA cells. Calphostin C, a PKC inhibitor, inhibited the TG-induced [Ca2+]i elevation dose-dependently (0.1-1 microM) and attenuated Mn2+ entry. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, potentiated the elevation of [Ca2+]i and enhanced Mn2+ entry by TG treatment. These results suggest that PKC may modulate capacitative Ca2+ entry in BA cells. In the presence of extracellular Ca2+, TG enhanced cortisol production in BA cells. Calphostin C attenuated the TG-induced steroidogenesis dose-dependently (0.25-1 microM). PMA enhanced the steroidogenesis dose-dependently (1-100 nM). These results suggested that PKC may have a modulatory effect on the capacitative Ca2+ entry that links to steroidogenesis in BA cells.     

Effect of fluoxetine on intracellular Ca2+ levels in bladder female transitional carcinoma (BFTC) cells.

The effect of the antidepressant fluoxetine on Ca2+ signaling in cultured cells was largely unknown. The effect of various concentrations of fluoxetine on [Ca 2+] i in populations of bladder female transitional cancer (BFTC) cells was evaluated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca 2+] i concentration dependently (20-100 microM) with an EC50 value of 30 microM. The response was inhibited by 50-60% on extracellular Ca2+ removal. In Ca2+ -free medium, pretreatment with 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) abolished 50 microM fluoxetine-induced Ca2+ release; whereas pretreatment with fluoxetine did not alter the thapsigargin-induced Ca2+ response. Addition of 3 mM Ca2+ increased [Ca 2+] i after pretreatment with 50 microM fluoxetine in Ca2+ -free medium, suggestive of fluoxetine-induced capacitative Ca2+ entry. Suppression of inositol 1,4,5-trisphosphate formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 50 microM fluoxetine-induced Ca2+ release. Collectively, this study shows that fluoxetine increased [Ca 2+] i in bladder cancer cells in a concentration-dependent fashion, by releasing Ca2+ from thapsigargin-sensitive Ca2+ stores in an IP3-independent manner, and by inducing Ca2+ influx from extracellular medium.     

Gossypol, a component in cottonseed, induced increases in cytosolic Ca2+ levels in Chang liver cells.

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.