Evidence for a discrete UTP receptor in cardiac endothelial cells.

1. We have examined the effects of various purine and pyrimidine nucleotides upon cells cultured from guinea-pig cardiac endothelium (CEC), and find the P2Y-agonist 2-methylthioadenosine triphosphate (2MeSATP) to be a potent (EC50 = 85 +/- 10.2 nM) stimulator of increase in intracellular calcium concentrations, while uridine 5'-triphosphate (UTP) and adenosine 5'-triphosphate (ATP) are less potent but equipotent with one another (EC50s = 2.1 +/- 0.3 and 1.8 +/- 0.2 microM, respectively). 2. While the P2Y receptor exhibited rapid homologous desensitization, this had no effect upon subsequent responsiveness of CEC to either ATP or UTP. Effects of maximal concentrations of ATP and UTP were not only additive, but did not cross-desensitize. Responses to UTP (but not to ATP or 2MeSATP) were blocked by treatment with pertussis toxin (PTX); all three nucleotides appeared to liberate calcium from an intracellular pool. 3. Suramin (30 microM) significantly (P < 0.05) increased the EC50 for ATP-dependent increases in intracellular calcium (5.3 +/- 2.2 microM vs. 2.0 +/- 0.9 microM in the absence of suramin), while it completely blocked the response to 2MeSATP. Suramin had no effect upon responses to UTP at concentrations of 100 microM. 4. We conclude that in addition to the P2Y and P2U subtypes of the ATP receptor, an additional receptor responsive to UTP but exhibiting no affinity for purine nucleotides is present in CEC; this "pyrimidine receptor' liberates intracellular calcium via a G-protein, and may partly mediate the contractile response to UTP in the coronary vasculature.     

4-aminopyridine affects rat arterial smooth muscle BK(Ca) currents by changing intracellular pH

The hypothesis whether or not 4-AP can affect vascular smooth muscle BK(Ca) currents was tested using the patch-clamp technique, pH- and calcium-fluorimetry, and freshly isolated rat arterial smooth muscle cells. Application of 4-AP reversibly inhibited BK(Ca) currents at an intracellular calcium ([Ca](i)) of 250 nM with a half-block of 2. 5 mM at +50 mV. The presence of 2 microM thapsigargin, 10 microM heparin, and 10 microM ryanodine did not alter the effect of 4-AP on BK(Ca) currents at [Ca](i) 250 nM. At [Ca](i)<100 nM 4-AP did not inhibit BK(Ca) currents. Application of 4-AP to the intracellular or extracellular side of excised BK(Ca) channels did not alter channel activity or channel amplitude. Replacement of the pH-sensitive calcium buffer EGTA by the pH-insensitive calcium buffer BAPTA in the intracellular solution turned the 4-AP-induced inhibition of BK(Ca) currents into a stimulation at [Ca](i) 250 nM. Application of 4-AP to single cells increased intracellular pH, which was accompanied by a reduction of [Ca](i) in EGTA-loaded cells and a stable [Ca](i) in BAPTA-loaded cells. Thus, these results suggest that in isolated vascular smooth muscle cells at [Ca](i)>100 nM 4-AP affects BK(Ca) currents via an alteration of intracellular pH.     

Mechanism of endothelin-induced contraction in guinea-pig trachea: comparison with rat aorta.

1. Endothelin (1 nM-0.3 microM) produced a concentration-dependent contraction of guinea-pig epithelium-containing (intact) trachea (EC50 = 30.9 nM). Endothelin was a less potent agonist than leukotriene D4 (LTD4; EC50 = 0.77 nM), but was more potent than carbachol (EC50 = 0.15 microM) or substance P (EC50 = 1.4 microM). Endothelin was a more potent contractile agent in rat endothelium-denuded aorta (EC50 = 2.1 nM) than in guinea-pig trachea. 2. Endothelin-induced contraction in guinea-pig trachea was unaffected by mepyramine (10 microM), atropine (1 microM), SK&F 104353 (10 microM), a leukotriene receptor antagonist, or SQ 29,548 (1 microM), a thromboxane receptor antagonist. The contraction produced by 0.3 microM endothelin was potentiated by cyclo-oxygenase inhibition with 5 microM indomethacin. 3. Nicardipine (0.01 or 0.1 microM) or incubation in calcium-free medium +0.1 mM EGTA for 30 min had a relatively minor or no effect on endothelin concentration-response curves in guinea-pig intact trachea, but markedly inhibited responses produced by endothelin in endothelium-denuded aorta of the rat. Increasing the EGTA concentration in calcium-free medium to 1 mM abolished endothelin-induced contraction in guinea-pig trachea. 4. In guinea-pig trachea, ryanodine (10 microM) produced a 2.1 fold shift to the right of endothelin concentration-response curves and reduced the maximum response elicited by 0.3 microM endothelin. 5. Staurosporine (0.01 microM and 0.1 microM), a protein kinase C inhibitor, was without effect on endothelin- or carbachol-induced contraction in guinea-pig trachea, but markedly inhibited the response produced by endothelin in rat aorta. 6. Endothelin (3 nM-0.3 microM) produced a concentration-dependent stimulation of phosphatidylinositol (PI) turnover in guinea-pig intact trachea, with an EC50 value of 45.9 nM. 7. Removal of the epithlium markedly potentiated endothelin-induced contraction in guinea-pig trachea, producing a 4.7 fold leftward shift in endothelin concentration-response curves and an increase in the contractile response elicited by 0.3 microM endothelin. 8. These data indicate that endothelin is a potent agonist in guinea-pig trachea whose response is markedly enhanced by removal of the airway epithelium. Endothelin-induced contraction is not mediated to a marked extent by calcium influx via dihydropyridine-sensitive calcium channels and does not involve the release of histamine, acetylcholine, leukotrienes or thromboxane. Rather, endothelin appears to produce contraction of guinea-pig trachea via a direct action which involves stimulation of PI turnover and utilization of calcium from intracellular stores and, also, calcium influx via a pathway that is not sensitive to dihydropyridine calcium channel inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for P2Y1, P2Y2, P2Y6 and atypical UTP-sensitive receptors coupled to rises in intracellular calcium in mouse cultured superior cervical ganglion neurons and glia

1 P2Y receptors are expressed in the nervous system and are involved in calcium signalling in neurons and glia. In the superior cervical ganglion (SCG), RT-PCR analysis indicated the presence of P2Y(1,2&6) receptors. Rises in intracellular calcium in response to P2Y receptor stimulation were determined from adult mouse cultured SCG neurons and glia. 2 ADP evoked suramin (100 microM)- and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM)-sensitive rises in intracellular calcium in approximately 80% of SCG neurons (EC50 approximately 20 microM). ADP-evoked responses were abolished in neurons from P2Y1 receptor-deficient mice (responses to UTP were unaffected). 3 The pyrimidines UTP (EC50 approximately 85 microM) and UDP (EC50>90 microM) evoked PPADS- and suramin-sensitive responses in approximately 70 and approximately 20% of SCG neurons, respectively. 4 In SCG glial cells, ADP (EC50 approximately 30 microM) evoked calcium responses in approximately 50% of glia. These were suramin and PPADS sensitive and essentially abolished in SCG glial cells cultured from adult P2Y1 receptor-deficient mice. 5 UTP (EC50 approximately 25 microM) and UDP (EC50>200 microM) evoked suramin- and pyridoxalphosphate-6-azophenyl-2',5'-disulphonate-sensitive rises in calcium in approximately 60 and 20% SCG glial cells, respectively. 6 These results indicate the presence of several P2Y receptors coupled to an increase in intracellular calcium in the SCG: ADP-sensitive P2Y1 receptors and UDP-sensitive P2Y6 receptors in SCG neurons and glial cells, a novel UTP-sensitive P2Y receptor in SCG neurons and UTP- and ATP-sensitive P2Y2 receptors in SCG glia.     

Effect of calmidazolium analogs on calcium influx in HL-60 cells

The structure-activity relationships of calmidazolium analogs with respect to intracellular calcium levels were investigated in HL-60 cells. Quaternized derivatives of miconazole and clotrimazole, known inhibitors of store-operated calcium (SOC) channels, were synthesized. The quaternary N-methyl derivatives of miconazole (3) and clotrimazole (6) had no effect on intracellular calcium levels, alone or after elevation of calcium induced by ATP. Calmidazolium alone induced a large increase in intracellular calcium levels in HL-60 cells (EC(50) 3 microM). Similar effects were observed for miconazole derivatives 1 (EC(50) 15 microM) and 2 (EC(50) 10 microM), wherein the diphenylmethyl group in calmidazolium was replaced by a 3,5-difluorobenzyl or cyclohexylmethyl group, respectively. The analogous clotrimazole derivatives 4 and 5 had no effect on intracellular calcium levels. The elevation of calcium levels by calmidazolium, 1, and 2 appears to be comprised of a calcium release component from inositol trisphosphate (IP(3))-sensitive stores followed by a large calcium influx component. Calcium influx was greater than that normally observed due to depletion of IP(3)-sensitive calcium stores and activation of SOC channels. In addition, only a small component of the calmidazolium-elicited influx was inhibited by the SOC channel blocker miconazole. Thus, certain quaternized imidazoles substituted with large residues at both nitrogens of the imidazole ring caused both release and influx of calcium, the latter in part through SOC channels but mainly through an undefined cationic channel. Quaternized imidazoles, unlike the parent nonquaternary imidazole miconazole, did not block SOC channels. Inhibitory effects on calmodulin-activated phosphodiesterase did not correlate with effects on calcium release and influx.     

Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells

Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases. The present study was performed to evaluate the inhibitory effect of fangchinoline, a bis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline (1 and 5 microM) inhibited glutamate (1 mM), N-methyl-D-aspartate (NMDA; 1 mM) and kainate (100 microM)-induced neuronal cell death which was measured by trypan blue exclusion test. Fangchinoline (1 and 5 microM) inhibited glutamate release into medium induced by NMDA (1 mM) and kainate (100 microM), which was measured by HPLC. And fangchinoline (5 microM) inhibited glutamate (1 mM)-induced elevation of intracellular calcium concentration. These results suggest that inhibition of Ca2+ influx by fangchinoline may contribute to the beneficial effects on neurodegenerative effect of glutamate in pathophysiological conditions.     

2'-C-methylcytidine as a potent and selective inhibitor of the replication of foot-and-mouth disease virus

We report on the potent and selective in vitro antiviral activity of 2'-C-methylcytidine (2'-C-MetCyt) against foot-and-mouth disease virus (FMDV). FMDV belongs to the Picornaviridae and has the potential to cause devastating epidemics in livestock. The 50% and 90% effective concentrations (EC50 and EC90) for inhibition of the FMDV-induced cytopathic effect (CPE) formation were 6.4+/-3.8 and 10.8+/-5.4 microM. Comparable EC50 values for inhibition of viral RNA synthesis were observed. Treatment of FMDV-infected BHK-21 cells with 77 microM 2'-C-MetCyt resulted in a (1.6-3.2)x10(3)-fold reduction of infectious virus yield. Time-of-drug addition experiments suggest that 2'-C-MetCyt interacts with viral replication at a time point that coincides with the onset of intracellular viral RNA synthesis. In contrast to emergency vaccination, a potent and selective antiviral agent may provide almost immediate (prophylactic/therapeutic) protection against infection and thus constitute an important alternative/supplementary option to contain outbreaks such as those caused by FMDV.     

Inhibition of receptor-mediated calcium responses by corticotrophin-releasing hormone in the CATH.a cell line

A region of the brain believed to be important in the CNS response to stress is the locus coeruleus, the predominant site of noradrenergic cell bodies. Corticotrophin releasing hormone (CRH) is the primary hypothalamic releasing hormone responsible for the activation of the pituitary-adrenal axis in response to stress and, in this study, we employed a locus coeruleus-like cell line, CATH.a, to investigate the modulation of receptor signalling pathways by CRH. Pituitary adenylyl cyclase-activating polypeptide (PACAP) (10 nM), vasoactive intestinal peptide (VIP) (1 microM) and carbachol (1 mM) produced transient increases in intracellular [Ca2+]. The inhibition of the carbachol (1 mM) response by CRH was concentration-dependent (EC50 = 154 +/- 1.8 nM). Calcium responses to sub-maximally effective concentrations of PACAP (5 nM), VIP (400 nM) and carbachol (1 mM) were abolished by prior exposure to CRH (1 microM). At the concentrations employed, CRH and VIP both substantially increased intracellular [3H]-cyclic AMP accumulation. The adenylyl cyclase activator forskolin (10 microM) was also effective at eliminating the agonist-induced calcium responses. Incubation with the cell permeant cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) (1 mM), an activator of protein kinase A (PKA), for 12 min prior to agonist exposure similarly abolished the intracellular calcium response to carbachol. Carbachol increased [3H]-inositol phosphate ([3H]-IP) accumulation to a maximum of 2.4 +/- 0.11-fold basal (EC50 = 6.75 +/- 0.26 microM). PACAP produced a much greater accumulation (19.9 +/- 2.1 fold basal; EC50 = 24 nM). In the presence of forskolin (10 microM), neither carbachol- nor PACAP-induced [3H]-IP accumulation was significantly different from in its absence. These results demonstrate that CRH inhibits receptor-mediated intracellular calcium responses in a locus coeruleus-like cell line possibly via activation of PKA. This modulation could be important in controlling neuronal function in vivo in stressful situations in which the levels of CRH are increased in the locus coeruleus.     

Activities of caffeine, theophylline, and enprofylline analogs as tracheal relaxants

A variety of xanthines cause tracheal relaxation, an activity predictive of antiasthmatic potential. Structural analogs of caffeine, theophylline, and enprofylline were examined for their abilities to relax carbamylcholine-stimulated guinea pig trachea in vitro. All caffeine analogs tested were more potent than caffeine (EC50 = 551 +/- 81 microM) except the 8-p-sulfophenyl analog. 1,3,7-Tripropylxanthine and 1,3,7-tripropargylxanthine were among the more potent analogs with EC50 values of 12 +/- 1.3 and 65 +/- 11 microM respectively. Increasing the polarity at the 1- or 3-position by substituting a propargyl group for an n-propyl group decreased relaxant activity, an effect not observed at the 7-position. The 8-cyclohexyl-, 8-cyclopentyl- and 8-phenyl-derivatives of caffeine were relatively potent (EC 50 = approximately 75 microM). The theophylline analog 1,3-di-n-propylxanthine was approximately two times more active than theophylline (EC50 = 162 +/- 17 microM). 3-Isobutyl-1-methylxanthine (EC50 = 7.1 +/- 1.8 microM) and 1-isoamyl-3-isobutylxanthine (EC50 = 37 +/- 4.2 microM) were among the most potent tracheal relaxants. The 8-substituted theophylline analogs were weak or inactive relaxants except for 8-cyclopentyl- and 8-cyclohexyltheophylline, which were more potent or as potent as theophylline. In contrast to enprofylline (EC50 = 56 +/- 9 microM), 8-substituted enprofylline analogs were weak or inactive as relaxants with the exception of the 8-cyclohexyl analog. The potency of xanthines as tracheal relaxants was unrelated to potency as adenosine receptor antagonists and may reflect activity as phosphodiesterase inhibitors.     

Excitotoxic profiles of novel, low-affinity kainate receptor agonists in primary cultures of murine cerebellar granule cells.

The involvement of low-affinity kainate (KA) receptors in neuronal injury was investigated by employing a variety of agonists active at GluR5-7. Their excitotoxic profiles were determined in primary cultures of cerebellar granule cells, which abundantly expressed low-affinity KA receptors, and in the absence of any AMPA receptor-mediated neurotoxicity. Neurotoxicity induced by these compounds was analysed by phase contrast microscopy, a cell viability assay, the TUNEL technique (apoptosis), and by employing propidium iodide (PI; necrosis). All agonists induced concentration-dependent neurotoxicity, with rank order (EC(50) values; microM): (S)-iodowillardiine (IW) 0.2>(2S,4R)-4-methylglutamate (4-MG) 36>(2S,4R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) 46>KA 74>(RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4yl)propanoic acid (ATPA) 88. IW exposure resulted in apoptosis at lower concentrations (<30 microM) and necrosis at higher concentrations, both of which were attenuated by CNQX (50 microM), but not MK-801 (10 microM). ATPA-mediated neurotoxicity was purely apoptotic and was attenuated by the non-NMDA receptor antagonists. Both IW and ATPA induced injury with the morphological characteristics of apoptosis shown by the presence of TUNEL-positive neurones. LY339434-mediated neuronal injury was only attenuated by MK-801 and was necrotic in nature. Similarly, 4-MG (>30 microM) exposure caused necrosis that was partially attenuated by MK-801 (10 microM) and CNQX (50 microM). The patterns of neurotoxicity possessed a complex pharmacological profile, demonstrated an apoptotic-necrotic continuum and were inconsistent with past findings, further outlining the importance of characterizing novel compounds at native receptors. ATPA and to a lesser extent IW appear to be suitable drugs for low-affinity KA receptors. Since toxicity-mediated by low-affinity KA receptors seem likely to contribute to neurodegenerative conditions, our study importantly examines the excitotoxic profile of these novel agonists.     

4-Aminopyridine-induced increase in basal and stimulation-evoked [3H]-NA release in slices from rat hippocampus: Ca2+ sensitivity and presynaptic control.

1. We have examined the mechanisms by which the K(+)-channel blocker 4-aminopyridine (4-AP) can dose-dependently increase both basal [3H]-noradrenaline ([3H]-NA) release and the [3H]-NA release evoked by electrical stimulation, but not the release of [3H]-acetylcholine ([3H]-ACh), from slices of rat hippocampus. 2. Both the electrically evoked and the 4-AP-induced release were blocked by tetrodotoxin (TTX) (3 microM). The Ca(2+)-dependence of the 4-AP-induced release (EC50 0.15 mM) was, however, different from that of the electrically evoked [3H]-NA release (EC50 0.76 mM). 3. The 4-AP-induced release could be inhibited by CdCl2(10 microM) and omega-conotoxin (30 nM), but not by nifedipine (1 microM). 4. Transmitter release evoked by 100 microM 4-AP could be blocked by the alpha 2-adrenoceptor agonist, UK 14,304 (0.1 microM) and by the A1-receptor agonist R-N6-phenylisopropyl adenosine (R-PIA, 1 microM) and increased by the alpha 2-adrenoceptor antagonist, yohimbine (1 microM), both in 0.25 and 1.3 mM Ca(2+)-containing medium. By contrast, the effect of alpha 2-adrenoceptor agonist and antagonists on transmitter release evoked by electrical stimulation was markedly reduced in the presence of 4-AP (100 microM). 5. The results suggest that 4-AP can depolarize some nerve endings in the central nervous system, leading to transmitter release that is dependent on nerve impulses and Ca2+. Furthermore, the fact that alpha 2-receptors and adenosine A1 receptor agonists can influence the release of NA evoked by 4-AP suggests that these drugs may have actions that are independent of blockade of aminopyridine-sensitive K(+)-channels.     

Stereochemistry and molecular pharmacology of (S)-thio-ATPA, a new potent and selective GluR5 agonist

(RS)-2-Amino-3-(5-tert-butyl-3-hydroxy-4-isothiazolyl)propionic acid (thio-ATPA), a 3-isothiazolol analogue of (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), has previously been shown to be a relatively weak AMPA receptor agonist at native (S)-glutamic acid ((S)-Glu) receptors (EC(50)=14 microM), comparable in potency with ATPA (EC(50)=34 microM). Recent findings, that (S)-ATPA is a potent (EC(50)=0.48 microM) and selective agonist at homomerically expressed ionotropic GluR5, prompted us to resolve thio-ATPA using chiral chromatography and pharmacologically characterize the two enantiomers at native as well as cloned ionotropic glutamate receptors. The enantiomers, (S)- and (R)-thio-ATPA, were obtained in high enantiomeric excess, and their absolute stereochemistry established by an X-ray crystallographic analysis. Electrophysiologically, the two enantiomers were evaluated in the rat cortical wedge preparation, and the S-enantiomer was found to be an AMPA receptor agonist (EC(50)=8.7 microM) twice as potent as the racemate, whereas the R-enantiomer was devoid of activity. In accordance with this, (S)-thio-ATPA proved to be an agonist at homomerically expressed recombinant AMPA receptors (GluR1o, GluR3o, and GluR4o) with EC(50) values of 5, 32 and 20 microM, respectively, producing maximal steady state currents of 78--168% of those maximally evoked by kainic acid, and 120-1600% of those maximally evoked by (S)-ATPA. At homomerically expressed GluR5, (S)-thio-ATPA was found to be a potent agonist (EC(50)=0.10 microM), thus being approximately five times more potent than (S)-ATPA. (R)-Thio-ATPA induced saturating currents with an estimated EC(50) value of 10 microM, most likely due to a contamination with (S)-thio-ATPA. At heteromerically expressed GluR6+KA2 receptors, (S)-thio-ATPA showed relatively weak agonistic properties (EC(50)=4.9 microM). Thus, (S)-thio-ATPA has been shown to be a very potent agonist at GluR5, and may be a valuable tool for the investigation of desensitization properties of AMPA receptors.     

Effects of BRL 38227, sodium nitroprusside and verapamil on collateral perfusion following acute arterial occlusion in the rabbit isolated ear.

1. We have used an isolated, buffer-perfused, rabbit ear model of acute arterial occlusion to investigate the effects of the nitrovasodilator sodium nitroprusside, the potassium channel activator BRL 38227 (the active (-)-enantiomer of cromakalim) and the calcium antagonist, verapamil, on collateral perfusion in the absence of pharmacological tone. 2. Verapamil was the most potent vasodilator (EC50 = 72.6 +/- 32.0 nM) of 5-hydroxytryptamine/histamine-induced tone in the rabbit isolated perfused ear. Sodium nitroprusside and BRL 38227 were less potent with respective EC50 values of 488 +/- 75 nM and 296 +/- 40 nM. Following inhibition of endothelium-derived relaxing factor (EDRF) synthesis, the potency of BRL 38227 was significantly (P less than 0.001) increased with an EC50 of 55.6 +/- 5.0 nM. 3. BRL 38227 at 500 nM and 3 microM induced substantial increases in collateral perfusion following arterial ligation in the absence of pharmacological tone compared to control. Furthermore 3 microM BRL 38227 completely reversed the attenuation of collateral perfusion which followed inhibition of EDRF synthesis with 100 microM NG-nitro-L-arginine methyl ester (L-NAME). 4. Sodium nitroprusside (500 nM and 3 microM) induced modest improvements in collateral perfusion in the early stages after arterial occlusion. 5. Verapamil did not influence collateral perfusion at either of the concentrations used (50 nM and 3 microM), even though it was a potent vasodilator. 6. The results of this study indicate that BRL 38227, and to a much lesser extent sodium nitroprusside, selectively improve collateral perfusion following arterial occlusion, even in the presence of effects of EDRF on acute collateralization, while verapamil has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1)

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC(50) values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90+/-0.20 microM; human=1.90+/-0.30 microM: CHO cells: rat=0.20+/-0.06 microM; human=0.19+/-0.08 microM). In CHO cell lines co-expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC(50) values for capsaicin-induced responses were similar in both cell lines (rat=0.35+/-0.06 microM, human=0.53+/-0.03 microM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC(50) pH 5.49+/-0.04 and pH 5.78+/-0.09, respectively). The threshold for heat activation was identical (42 degrees C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC(50) between 3 and 10 microM) but was virtually inactive at the human VR1 (EC(50)>10 microM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.     

Apoptosis of cultured astrocytes induced by the copper and neocuproine complex through oxidative stress and JNK activation.

Astrocytes play a critical neurotrophic and neuroprotective role in the brain, and improper function of these cells may contribute to the onset of neurodegenerative diseases. Because astrocytes are known to be enriched with Cu chaperone proteins, it is important to understand the factors that may lead to cytotoxic effects of Cu on astrocytes. In this report, we demonstrated a dramatic potentiating effect of neocuproine (NCP), a membrane permeable metal chelator, on Cu, but not Fe or Pb, in inducing apoptosis of cultured astrocytes. It was estimated that individually, CuCl2 and NCP only weakly exhibited cytotoxic effects on astrocytes, with EC50 of 180 and 600 microM, respectively. However, NCP at a nontoxic concentration of 10 microM markedly reduced EC50 of Cu to 0.35 microM (physiological concentration) and Cu (10 microM) reduced EC50 of NCP down to 0.06 microM. The mechanisms underlying these dramatic potentiation effects are elucidated. NCP increased the intracellular concentration of Cu in astrocytes and a nonpermeable Cu chelator, bathocuproine disulfonate was able to abolish all of the apoptotic signaling. Cell death was determined to be via apoptosis due to increased reactive oxygen species production, mitochondrial dysfunction, depletion of glutathione and adenosine triphosphate, cytochrome c release, c-Jun N-terminal kinase, and caspase-3 activation, and poly-ADP-ribose polymerase degradation. This finding, coupled with our previous reports, suggests that metal chelators (NCP, dithiocarbamate and disulfiram) should be cautiously used as they may potentiate a cytotoxic effect of endogenous Cu on astrocytes. Their clinical implications in the etiology of neurodegenerative diseases deserve further investigation.     

Muscarinic receptors coupled to phosphoinositide hydrolysis and elevated cytosolic calcium in a human neuroblastoma cell line SK-N-SH.

1. The effects of the muscarinic agonist carbachol on phosphoinositide metabolism and its relationship to alteration of intracellular calcium were examined in SK-N-SH human neuroblastoma cells. Muscarinic receptors on these cells are coupled to phospholipase C and the myo [2-3H]-inositol phosphates resulting from receptor activation of cells labelled with [3H]-inositol accumulate rapidly. The breakdown of both inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) is sensitive to lithium with inhibition of the latter only observed at higher concentrations of this ion. 2. Use of the calcium indicator dye Fura 2 revealed that carbachol stimulates a biphasic increase in intracellular calcium. 3. Carbachol was able to stimulate both [3H]-inositol phosphate production and intracellular calcium levels with respective EC50 values of 15.9 +/- 1.0 microM and 10.7 +/- 3.2 microM, indicating that no amplification occurs between these steps in the signal transduction pathway. 4. Inositol 1,4,5 trisphosphate (Ins(1,4,5)P3) released 45Ca2+ in a stereospecific and dose-related manner from intracellular stores of permeabilised cells. 5. These results suggest that this cell line may represent a useful model system to investigate receptor-mediated phosphoinositide metabolism and calcium homeostasis.     

Comparison of the vasorelaxing effect of cromakalim and the new inodilator, levosimendan, in human isolated portal vein

In the present study the vasorelaxing capacity of cromakalim, an ATP-sensitive potassium-channel (KATP channel) activator, and that of levosimendan, a new positive inotropic and vasodilating drug with calcium sensitizing and potassium-channel-activating properties, were compared in human isolated portal vein. Based on the 50% effective concentrations (EC50), levosimendan was found to be about 16-fold more potent (EC50 = 0.281+/-0.03 microM) as a relaxing agent than cromakalim (EC50 = 4.53+/-0.12 microM) in noradrenaline-precontracted portal venous preparations. Glibenclamide, the known inhibitor of KATP channels, was able to prevent the cromakalim-induced venodilation completely. Glibenclamide (15 microM) decreased the quasi-maximal effect of levosimendan (at 1.27 microm by about 60%) and also the effects of those submicromolar concentrations of the inodilator (at 0.1 microM by 23%, at 0.3 microM by 27% and at 0.7 microM by 19%, on average) which were therapeutically effective in preliminary human studies. These findings indicate that, in the human portal vein, both cromakalim and levosimendan are powerful vasorelaxants and that a considerable part of the relaxing effect induced by levosimendan is of cromakalim type.     

Synthesis and antiviral activity of helioxanthin analogues

A series of natural product analogues based on helioxanthin (2), with particular attention to modification of the lactone ring and methylenedioxy group, were synthesized and evaluated for their antiviral activities. Among them, lactam derivative 18 and helioxanthin cyclic hydrazide 28 exhibited significant in vitro antiviral activity against hepatitis B virus (EC(50) = 0.08 and 0.03 microM, respectively). Compound 18 showed the most potent antiviral activity against hepatitis C virus (55% inhibition at 1.0 microM). Compound 12, an acid-hydrolyzed product of helioxanthin cyclic imide derivative 9, was found to exhibit broad-spectrum antiviral activity against hepatitis B virus (EC(50) = 0.8 microM), herpes simplex virus type 1 (EC(50) = 0.15 microM) and type 2 (EC(50) < 0.1 microM), Epstein-Barr virus (EC(50) = 9.0 microM), and cytomegalovirus (EC(50) = 0.45 microM). Helioxanthin lactam derivative 18 also showed marked inhibition of herpes simplex virus type 1 (EC(50) = 0.29 microM) and type 2 (EC(50) = 0.16 microM). The cyclic hydrazide derivative of helioxanthin 28 and its brominated product 42 exhibited moderately potent activities against human immunodeficiency virus (EC(50) = 2.7 and 2.5 microM, respectively). Collectively, these molecules represent a novel set of antiviral compounds with unique structural features.     

Role of prostanoids in the contraction induced by a tachykinin NK2 receptor agonist in the hamster urinary bladder

In isolated strips of the hamster urinary bladder the selective tachykinin NK2 receptor agonist [betaAla8]NKA(4-10) induced a concentration-dependent (1 nM-10 microM) contraction (EC50 104 nM) associated with significant release of prostaglandin E2 (PGE2, 50+/-17 pg/mg tissue). In mucosa-free bladder strips [betaAla8]NKA(4-10) was as potent as in the presence of mucosa (EC50 46 nM), although the evoked PGE2 release was significantly less than in controls (6+/-1.7 pg/mg tissue). Dexketoprofen (10 microM) produced a significant but limited rightward shift of the concentration/response curve to [betaAla8]NKA(4-10) both in the presence and absence of the mucosal layer: the EC50 for [betaAla8]NKA(4-10) was increased five- and threefold, respectively. The evoked PGE2 release was abolished in both cases. The selective tachykinin NK2 receptor antagonist, nepadutant (10 nM-1 microM) produced a concentration-dependent and even inhibition of both contraction and PGE2 release induced by [betaAla8]NKA(4-10). The L-type calcium channel blocker, nifedipine (1 microM) and the non-selective cationic channel blocker SKF 96365 (30 microM) both inhibited the contractile response to [betaAla8]NKA(4-10) (89+/-2 and 83+/-2% inhibition, respectively). The evoked PGE2 release was not affected by nifedipine but was almost abolished by SKF 96365. Arachidonic acid (100 microM) induced a contractile response (5.9+/-0.7 mN) associated with a large production of PGE2 (383+/-78 pg/mg tissue). The contractile response to arachidonic acid was inhibited by both nifedipine (1 microM) and SKF 96365 (30 microM) (83+/-5 and 79+/-3% inhibition, respectively). The PGE2 production induced by arachidonic acid was markedly inhibited by SKF 96365 only (about 94% inhibition). Exogenous PGE2 contracted hamster bladder strips in the presence of dexketoprofen (EC50 1 microM) and strongly potentiated the contractile response to a submaximal concentration of [betaAla8]NKA(4-10). In anaesthetized hamsters the administration of [betaAla8]NKA(4-10) (10 nmol/kg, i.v.) produced a contractile response of the urinary bladder (13+/-0.4 mmHg) that was inhibited partly by dexketoprofen (25+/-3 and 35+/-4% inhibition for 0.2 and 2 mg/kg, i.v. dexketoprofen, respectively). We conclude that activation of tachykinin NK2 receptors determines prostanoid synthesis/release in the hamster urinary bladder and that this effect is largely ascribable to structures present in the bladder mucosa. Prostanoids generated following NK2 receptor activation amplify the direct contractile effect of NK2 receptor agonists. This latter response is largely due to activation of L-type calcium channels (nifedipine-sensitive) although this source of calcium apparently is not essential for activation of prostanoid synthesis.     

Neurokinin A receptors are coupled to calcium mobilization in transfected fibroblasts.

Neurokinin A (NKA) mediated a concentration dependent increase in the intracellular free Ca2+ concentration, [Ca2+]i, in B82 fibroblasts transfected with the neurokinin 2 (NK2) receptor. The EC50 value of this response was 24 nM. A selective NK2 antagonist, MEN 10207, at a concentration of 1 microM completely inhibited the [Ca2+]i rise to 0.1 microM NKA. These results suggest that activation of NK2 receptors expressed in the transfected fibroblasts are functionally coupled to intracellular calcium mobilization.