Sensitization of glutamate receptor-mediated pain behaviour via nerve growth factor-dependent phosphorylation of transient receptor potential V1 under inflammatory conditions.

BACKGROUND AND PURPOSE: Glutamate and metabotropic glutamate (mGlu) receptors on primary sensory neurons are crucial in modulating pain sensitivity. However, it is unclear how inflammation affects mGlu receptor-mediated nociceptive responses. We therefore investigated the effects of mGlu1/5 receptor agonists on pain-related behaviour during persistent inflammation and their underlying mechanisms. EXPERIMENTAL APPROACH: Effects of a mGlu1/5 receptor agonist on pain-related behaviour during inflammation was assessed in mice. Intracellular calcium responses, membrane current responses, and protein expression in primary sensory neurons were examined using cultured dorsal root ganglion (DRG) neurons, dissociated from wild-type and gene knockout mice. KEY RESULTS: Persistent inflammation induced by complete Freund's adjuvant increased the duration of mGlu1/5 receptor-mediated pain behaviour, which was antagonized by inhibition of nerve growth factor (NGF)-tropomyosin receptor kinase A (TrkA) signalling. Calcium imaging revealed that NGF treatment increased the number of cultured DRG neurons responding to mGlu1/5 receptor activation. Stimulation of mGlu1/5 receptors in NGF-treated DRG neurons induced inward currents through TRPV1 channels in association with PLC but not with IP3 receptors. NGF treatment also increased the number of neurons responding to a DAG analogue via TRPV1 channel activation. Furthermore, NGF up-regulated expression of TRPV1 and A-kinase anchoring protein 5 (AKAP5), resulting in increased AKAP5-dependent TRPV1 phosphorylation. AKAP5 knockout mice did not exhibit mGlu1/5 receptor-mediated excitation in NGF-treated DRG neurons or pain response facilitation under inflammatory conditions. CONCLUSIONS AND IMPLICATIONS: NGF augments glutamate- and mGlu1/5 receptor-mediated excitation of nociceptive neurons by AKAP5-dependent phosphorylation of TRPV1 channels, potentiating hypersensitivity to glutamate in inflamed tissues.

Impact of cigarette smoking on nitric oxide-sensitive and nitric oxide-insensitive soluble guanylate cyclase-mediated vascular tone regulation.

Cigarette smoking induces vascular endothelial dysfunction characterized by impaired nitric oxide (NO) bioavailability. There are two types of soluble guanylate cyclase (sGC), which is a cellular target of NO: NO-sensitive reduced form (the heme moiety with a ferrous iron) and NO-insensitive oxidized (the heme moiety with a ferric iron)/heme-free form. This study investigated the influence of cigarette smoking on NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation in organ chamber experiments with isolated rat and human arteries. The rats were subcutaneously administered phosphate-buffered saline (PBS), nicotine-free cigarette smoke extract (N(-)-CSE) or nicotine-containing cigarette smoke extract (N(+)-CSE) for 4 weeks. Plasma thiobarbituric acid reactive substance (TBARS) levels were higher in the N(+)-CSE group than those in the N(-)-CSE group, and TBARS levels for these groups were higher than those for the PBS group. In the aorta and the pulmonary artery in rats administered N(-)-CSE or N(+)-CSE, acetylcholine-induced relaxation was significantly impaired compared with that in rats administered PBS; there was no significant difference in the relaxation between the N(-)-CSE and N(+)-CSE groups. However, sodium nitroprusside (NO-sensitive sGC stimulant)- and BAY 60-2770 (NO-insensitive sGC stimulant)-induced relaxations were not different among the three groups, regardless of the vessel type. In addition, in the human gastroepiploic artery, the relaxant responses to these sGC-targeting drugs were identical between nonsmokers and smokers. These findings suggest that NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation functions normally even in blood vessels damaged by cigarette smoking.

Responsiveness of rat aorta and pulmonary artery to cGMP generators in the presence of thiol or heme oxidant.

This study investigated the effects of thiol and heme oxidants on responsiveness to cGMP generators in isolated rat aorta and pulmonary artery using an organ chamber. The nitric oxide (NO) donor sodium nitroprusside (SNP)-induced relaxation was impaired by exposure to the thiol oxidant diamide in both the aorta and the pulmonary artery, whereas the soluble guanylate cyclase (sGC) stimulator BAY 41-2272- or the sGC activator BAY 60-2770-induced relaxation was not affected. The impairment by diamide of SNP-induced aortic and pulmonary arterial relaxation was completely restored by post-treatment with the thiol reductant dithiothreitol. However, regardless of the vessel type, the relaxant response to SNP or BAY 41-2272 was impaired by exposure to the heme oxidant ODQ, whereas the response to BAY 60-2770 was enhanced. The ODQ-induced effects were reversed partially by post-treatment with the heme reductant dithionite. These findings indicate that thiol oxidation attenuates only the vascular responsiveness to NO donors and that heme oxidation attenuates the responsiveness to NO donors and sGC stimulators but augments that to sGC activators. Therefore, under oxidative stress, the order of usability of the vasodilators is suggested to be: NO donors < sGC stimulators < sGC activators.

Chronological Change of Vascular Reactivity to cGMP Generators in the Balloon-Injured Rat Carotid Artery.

BACKGROUND/AIMS: Soluble guanylate cyclase (sGC) exists as reduced, oxidized, and heme-free forms. Currently, it is unclear whether endovascular mechanical stenosis has an impact on vascular tone control by drugs targeting sGC, namely cGMP generators. METHODS: Pharmacological responses to acidified sodium nitrite (reduced sGC stimulant) and BAY 60-2770 (oxidized/heme-free sGC stimulant) were studied in balloon-injured rat carotid arteries at several time points. In addition, sGC expression was detected by immunohistochemistry. RESULTS: At 1 day after injury, acidified sodium nitrite-induced relaxation was attenuated in the injured artery, whereas BAY 60-2770-induced relaxation was augmented. Similar attenuation of response to acidified sodium nitrite was seen at 7 and 14 days after injury. On the other hand, the augmentation of response to BAY 60-2770 disappeared at 7 and 14 days after injury. At 1 day after injury, the immunohistochemical expression pattern of sGC in the smooth muscle layer of the injured artery was not different from that of the uninjured artery. However, in the injured artery, the intensity of sGC staining was weak at 7 and 14 days after injury. CONCLUSION: Balloon injury alters vascular responsiveness to cGMP generators, which seems to be associated with the form and/or expression of sGC.

Novel regulatory systems for acetylcholine release in rat striatum and anti-Alzheimer's disease drugs.

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [3 H]choline. Electrical stimulation-evoked pronounced [3 H]ACh release from cholinergic neurons. The estimated quantity of [3 H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre-synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M2 -selective antagonist AF-DX 116, partially inhibited by M3 -selective darifenacin, and minimally by M4 -selective PD 102807. Other subtype-selective antagonists had no effect at subtype-selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [3 H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre-synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [3 H]efflux was also enhanced by ACh uptake inhibitors (100 µM hemicholinium-3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre-synaptic M2 /M3 /M4 mAChRs, AChE and post-synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/.

Augmentation of Endogenous Acetylcholine Uptake and Cholinergic Facilitation of Hippocampal Long-Term Potentiation by Acetylcholinesterase Inhibition.

Hippocampal cholinergic activity enhances long-term potentiation (LTP) of synaptic transmission in intrahippocampal circuits and regulates cognitive function. We recently demonstrated intracellular distribution of functional M1-muscarinic acetylcholine receptors (mAChRs) and neuronal uptake of acetylcholine (ACh) in the central nervous system. Here we examined whether endogenous ACh acts on intracellular M1-mAChRs following its uptake and causes cholinergic facilitation of hippocampal LTP. ACh esterase (AChE) activities and [3H]ACh uptake was measured in rat hippocampal segments. LTP of evoked field excitatory postsynaptic potentials at CA1 synapses was induced by high frequency stimulation in hippocampal slices. Pretreatment with diisopropylfluorophosphate (DFP) irreversibly inhibited AChE, augmented ACh uptake, and significantly enhanced the LTP. This cholinergic facilitation was inhibited by pirenzepine, a membrane-permeable M1 antagonist, while only the early stage of cholinergic facilitation was inhibited by a membrane-impermeable M1 antagonist, muscarinic toxin 7. Tetraethylammonium (TEA) inhibited ACh uptake in hippocampal segments and selectively suppressed late stage cholinergic facilitation without changing the early stage. In contrast, LTP in DFP-untreated slices was not affected by the muscarinic antagonists and TEA. Carbachol (CCh; an AChE-resistant muscarinic agonist) competed with ACh for its uptake and produced cholinergic facilitation of LTP in DFP-untreated slices. The late stage of CCh-induced facilitation was also selectively inhibited by TEA. Our results suggest that when AChE is inactivated by inhibitors, LTP in hippocampal slices is significantly enhanced by endogenous ACh and that cholinergic facilitation is caused by direct activation of cell-surface M1-mAChRs and subsequent activation of intracellular M1-mAChRs after ACh uptake.

Regulation of synaptic acetylcholine concentrations by acetylcholine transport in rat striatal cholinergic transmission.

In addition to hydrolysis by acetylcholine esterase (AChE), acetylcholine (ACh) is also directly taken up into brain tissues. In this study, we examined whether the uptake of ACh is involved in the regulation of synaptic ACh concentrations. Superfusion experiments with rat striatal segments pre-incubated with [3 H]choline were performed using an ultra-mini superfusion vessel, which was developed to minimize superfusate retention within the vessel. Hemicholinium-3 (HC-3) at concentrations less than 1 µM, selectively inhibited the uptake of [3 H]choline by the high affinity-choline transporter 1 and had no effect on basal and electrically evoked [3 H]efflux in superfusion experiments. In contrast, HC-3 at higher concentrations, as well as tetraethylammonium (>10 µM), which inhibited the uptake of both [3 H]choline and [3 H]ACh, increased basal [3 H]overflow and potentiated electrically evoked [3 H]efflux. These effects of HC-3 and tetraethylammonium were also observed under conditions where tissue AChE was irreversibly inactivated by diisopropylfluorophosphate. Specifically, the potentiation of evoked [3 H]efflux was significantly higher in AChE-inactivated preparations and was attenuated by atropine. On the other hand, striatal segments pre-incubated with [3 H]ACh failed to increase [3 H]overflow in response to electrical stimulation. These results show that synaptic ACh concentrations are significantly regulated by the postsynaptic uptake of ACh, as well as by AChE hydrolysis and modulation of ACh release mediated through presynaptic muscarinic ACh receptors. In addition, these data suggest that the recycling of ACh-derived choline may be minor in cholinergic terminals. This study reveals a new mechanism of cholinergic transmission in the central nervous system.

TRPA1 Channels Modify TRPV1-Mediated Current Responses in Dorsal Root Ganglion Neurons.

The transient receptor potential vanilloid 1 (TRPV1) channel is highly expressed in a subset of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia of experimental animals, responsible for nociception. Many researches have revealed that some TRPV1-positive neurons co-express the transient receptor potential ankyrin 1 (TRPA1) channel whose activities are closely modulated by TRPV1 channel. However, it is less investigated whether the activities of TRPV1 channel are modulated by the presence of TRPA1 channel in primary sensory neurons. This study clarified the difference in electrophysiological responses induced by TRPV1 channel activation between TRPA1-positive and TRPA1-negative DRG. TRPV1 and TRPA1 channel activations were evoked by capsaicin (1 µM), a TRPV1 agonist, and allyl isothiocyanate (AITC; 500 µM), a TRPA1 agonist, respectively. Capsaicin perfusion for 15 s caused a large inward current without a desensitization phase at a membrane potential of -70 mV in AITC-insensitive DRG (current density; 29.6 ± 5.6 pA/pF, time constant of decay; 12.8 ± 1.8 s). The capsaicin-induced currents in AITC-sensitive DRG had a small current density (12.7 ± 2.9 pA/pF) with a large time constant of decay (24.3 ± 5.4 s). In calcium imaging with Fura-2, the peak response by capsaicin was small and duration reaching the peak response was long in AITC-sensitive neurons. These electrophysiological differences were completely eliminated by HC-030031, a TRPA1 antagonist, in an extracellular solution or 10 mM EGTA, a Ca2+ chelator, in an internal solution. Capsaicin perfusion for 120 s desensitized the inward currents after a transient peak. The decay during capsaicin perfusion was notably slow in AITC-sensitive DRG; ratio of capsaicin-induced current 60 s after the treatment per the peak current in AITC-sensitive neurons (78 ± 9%) was larger than that in AITC-insensitive neurons (48 ± 5%). The capsaicin-induced current in the desensitization phase was attenuated by HC-030031 in AITC-insensitive DRG. These results indicate that (1) TRPV1-mediated currents in TRPA1-positive neurons characterize small current densities with slow decay, which is caused by TRPA1 channel activities and intracellular Ca2+ mobilization and (2) desensitization of TRPV1-mediated current in TRPA1-positive neurons is apparently slow, due to appending TRPA1-mediated current.

Pharmacological evidence of specific acetylcholine transport in rat cerebral cortex and other brain regions.

Functional acetylcholine receptors (AChRs) were recently demonstrated to exist not only in the plasma membrane but also intracellularly in brain tissues. In order to activate intracellular AChRs, endogenous hydrophilic ACh must cross the plasma membrane. Here, we examined the pharmacological characteristics of this process, including whether it is mediated by active ACh uptake. When ACh esterase (AChE) was suppressed by diisopropylfluorophosphate, [3 H]ACh was effectively taken up into segments of rat cerebral cortex and other brain regions, in contrast to peripheral tissues such as liver and kidney. The uptake of [3 H]ACh in rat cerebral cortex was temperature-dependent, and the uptake capacity was comparable to that of [3 H]choline. However, [3 H]ACh uptake was inhibited by lower concentrations of ACh, carbachol, tetraethylammonium (TEA), compared with uptake of [3 H]choline. Uptake of [3 H]ACh was also inhibited by several organic cations, including choline, hemicholinium-3 (HC-3), quinidine, decynium 22, clonidine, diphenhydramine, but was little affected by some amino acids and biogenic amines, corticosterone, spermine, atropine, and tetrodotoxin. Unlike diisopropylfluorophosphate, several ACh esterase inhibitors, including drugs for Alzheimer's disease, such as donepezil, galantamine, and rivastigmine, also suppressed the uptake of [3 H]ACh, but not [3 H]choline. These results indicate that in the brain, ACh is specifically taken up through a unique transport system with different pharmacological properties from known organic cation transporters (OCTs), and suggest that this mechanism may be involved in intracellular cholinergic transmission in the brain.

Long-Term Activation of Group I Metabotropic Glutamate Receptors Increases Functional TRPV1-Expressing Neurons in Mouse Dorsal Root Ganglia.

Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.

Pharmacologically distinct phenotypes of α1B -adrenoceptors: variation in binding and functional affinities for antagonists.

BACKGROUND AND PURPOSE: The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α1B -adrenoceptor subtype in various tissue preparations and under various conditions. EXPERIMENTAL APPROACH: [(3) H]-prazosin binding to α1B -adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α1B -adrenoceptors. KEY RESULTS: In association and saturation-binding experiments with rat liver, binding affinity for [(3) H]-prazosin varied significantly between preparations (KD value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α1B -adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α1B -adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α1B -adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α1B -adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α1B -adrenoceptor profile. CONCLUSIONS AND IMPLICATIONS: In contrast to the consistent profile of recombinant α1B -adrenoceptors, the pharmacological profile of native α1B -adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional 'subtype' characterization, 'phenotype' pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy.

Intracellular localization of the M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization is mediated by a C-terminal tryptophan-based motif.

The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W(442) or I(445) with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxΦ, which is the canonical binding motif for the µ2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 µ2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-µ2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophan-based motif, which mediates constitutive internalization.

Role of Kampo medicine in integrative cancer therapy.

Clinical trials to date demonstrate that standard cancer treatments are currently the most efficient treatments for large numbers of cancer patients. Cancer treatments will increasingly require approaches that allow patients to live with cancer, by increasing their natural healing power and tumor immunity, as well as attenuating the progression of their cancers, instead of only attacking the cancer cells directly. Complementary and alternative medicine, including Kampo medicine, compensates for the drawbacks of western medicine by increasing patients' self-defense mechanisms. In Japan, clinicians who have studied both western medicine and Kampo treat cancer patients by fusing the two medical systems into a unitary one. The goal of the system is to assist the functional maintenance and recovery of the living body complex with the physical, mental, social, and spiritual balance, rather than addressing direct antitumor effects. In this review, we describe the usefulness of Kampo medicine, especially juzentaihoto, and outline the reports on evidence, in addition to the report on an attitudinal survey about the use of Kampo medicine in cancer treatment in Japan.

Significance of Kampo, traditional Japanese medicine, in supportive care of cancer patients.

The current standard treatment for cancer is a multidisciplinary therapy whereby various types of treatment are properly combined. Chemotherapy with multiple anticancer drugs is now common, and traditional, complementary, and alternative therapies are adopted as supportive measures. Medical care in Japan is distinguished by the ability for patients to access both Western and Kampo medical cares at the same time. There is a high degree of trust in the safety of Kampo therapies because they are practiced by medical doctors who are educated with fundamental diagnosis of Western medicine. Highly reliable clinical studies are being published, demonstrating that palliative or supportive care for cancer patients using Kampo preparations alleviates adverse effects of chemotherapy or radiotherapy. This paper reports the circumstances around cancer care in Japan where traditional therapeutic Kampo formulas are used for patients undergoing cancer treatment with cutting-edge chemotherapy, specifically to alleviate adverse effects of anticancer drugs.

Novel contribution of cell surface and intracellular M1-muscarinic acetylcholine receptors to synaptic plasticity in hippocampus.

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1-mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand-binding experiments with cell-permeable and -impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1-mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic-binding sites were abolished in M1-mAChR-gene-knockout mice. Activation of cell surface M1-mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long-term potentiation. On the other hand, activation of intracellular M1-mAChRs phosphorylated extracellular-regulated kinase 1/2 and gradually enhanced long-term potentiation. Our data thus demonstrate that M1-mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.

Effects of cyanamide, an aldehyde dehydrogenase type 2 inhibitor, on glyceryl trinitrate- and isosorbide dinitrate-induced vasodilation in rabbit excised aorta and in anesthetized whole animal.

The contribution of aldehyde dehydrogenase type 2 (ALDH2) to bioactivation of glyceryl trinitrate (GTN) and isosorbide dinitrate (ISDN) was systematically examined in excised rabbit aorta and anesthetized whole animal with cyanamide, an ALDH2 inhibitor. In excised aortic preparation, the degree of inhibition by cyanamide in GTN-induced vasorelaxation (concentration ratio, calculated as EC(50) in the presence of cyanamide/EC(50) in the absence of cyanamide; 5.61) was twice that in ISDN-induced relaxation (2.78). However, the degree of inhibition by cyanamide, as assessed by the dose ratio (as described above, but calculated with doses) in anesthetized rabbits was 2.29 in GTN-induced hypotension (assessed by area under the curve (AUC) of 50 mmHg·min) and 7.68 in ISDN-induced hypotension. Thus, the inhibitor was 3 times more potent in ISDN-induced hypotension, a finding in conflict with to that obtained in excised aortic preparation. The rate of increase in plasma nitrite (NO(2)(-)) concentration at certain hypotensive effect (50 mmHg·min of AUC) in the presence and absence of cyanamide (ΔNO(2)(-) ratio) was larger in ISDN-induced hypotension (15.01) than in GTN-induced hypotension (3.28). These results indicate that the bioactivation pathway(s) of GTN is ALDH2-dependent in aortic smooth muscle, while ADLH2-independent mechanism(s) largely take place in the whole body. In contrast, the activation mechanism(s) of ISDN is largely ALDH2-dependent in both aortic smooth muscle and whole body. Plasma NO(2)(-) may be derived from pathways other than the cyanamide-sensitive metabolic route.

Knockdown of stromal interaction molecule 1 (STIM1) suppresses store-operated calcium entry, cell proliferation and tumorigenicity in human epidermoid carcinoma A431 cells.

Store-operated calcium (Ca(2+)) entry (SOCE) is important for cellular activities such as gene transcription, cell cycle progression and proliferation in most non-excitable cells. Stromal interaction molecule 1 (STIM1), a newly identified Ca(2+)-sensing protein, monitors the depletion of endoplasmic reticulum (ER) Ca(2+) stores and activates store-operated Ca(2+) channels at the plasma membrane to induce SOCE. To investigate the possible roles of STIM1 in tumor growth in relation to SOCE, we established STIM1 knockdown (KD) clones of human epidermoid carcinoma A431 cells by RNA interference. Thapsigargin, an inhibitor of ER Ca(2+)-ATPase, -induced and phospholipase C-coupled receptor agonist-induced SOCEs were reduced in two STIM1 KD clones compared to a negative control clone. Re-expression of a KD-resistant full-length STIM1, but not a Ca(2+) release-activated Ca(2+) channel activation domain (CAD)-deleted STIM1 mutant, in the KD clone restored the amplitude of SOCE, suggesting the specificity of the STIM1 knockdown. The cell growth of the STIM1 KD clones was slower than that of the negative control clone. DNA synthesis assessed by BrdU incorporation, as well as EGF-stimulated EGF receptor activation, decreased in the STIM1 KD clones. Xenograft growth of the STIM1 KD clones was significantly retarded compared with that of the negative control. Cell migration was attenuated in the STIM1 KD clone and the STIM1 silencing effect was reversed by transient re-expression of the full-length STIM1 but not CAD-deletion mutant. These results indicate that STIM1 plays an important role in SOCE, cell-growth and tumorigenicity in human epidermoid carcinoma A431cells, suggesting the potential use of STIM1-targeting agents for treating epidermoid carcinoma.

Cyclooxygenase 2 and prostaglandin E2 regulate the attachment of calcium oxalate crystals to renal epithelial cells.

OBJECTIVE: To determine the roles of endogenous cyclooxygenase 2 and prostaglandin E(2) in crystal-cell binding, which is considered to be an important step in the development of intratubular nephrocalcinosis. METHODS: An expression plasmid for human cyclooxygenase 2 was introduced into Madin-Darby canine kidney cells using the lipofection method. Cyclooxygenase activity was measured using thin-layer chromatography, and the prostaglandin E(2) concentration was determined with an enzyme immunoassay. In addition, crystal attachment was evaluated with a liquid scintillation counter using [(14)C] calcium oxalate monohydrate crystals, and immunohistochemistry and an enzyme immunoassay were used to analyze and quantify the expression of hyaluronan, a crystal-binding molecule. RESULTS: Cyclooxygenase 2-overexpressing Madin-Darby canine kidney cells produced about 10-fold more prostaglandin E(2) than wild-type Madin-Darby canine kidney cells, and their hyaluronan production was also upregulated. The attachment of calcium oxalate monohydrate crystals to cyclooxygenase 2-overexpressing Madin-Darby canine kidney cells was significantly reduced compared with their attachment to wild-type and mock-transfected Madin-Darby canine kidney cells. Pre-incubation of the cyclooxygenase 2-overexpressing cells, as well as the mock-transfected and wild-type cells with the cyclooxygenase 2 selective inhibitor etodolac, increased the cellular attachment of calcium oxalate monohydrate crystals in a dose-dependent manner. CONCLUSIONS: These findings suggest that cyclooxygenase 2 expression and the resultant increase in endogenous prostaglandin E(2), leading to increased hyaluronan production, help to prevent nephrocalcinosis by inhibiting the attachment of calcium oxalate monohydrate crystals to the surface of renal epithelial cells.

Role of plasma S-nitrosothiols in regulation of blood pressure in anesthetized rabbits with special references to hypotensive effects of acetylcholine and nitrovasodilators.

The regulatory role of plasma nitrosothiols (R-SNOs) under steady-state conditions and their possible contribution to pharmacological vasodilation were systematically examined in anesthetized rabbits. Nitrosocystein (Cys-NO), S-nitrosoglutathione (G-SNO), and S-nitrosoalbumin (Alb-SNO) were determined by HPLC-Saville's method with respective sensitivities of 1, 1, and 5 nM. These R-SNOs were not detected under steady-state conditions even in the presence of N-ethylmaleimide, a thiol protective agent used to prevent transnitrosation of R-SNOs. Development of plasma Alb-SNO below 300 nM was observed after intravenous injection (i.v.) of nitric oxide (NO) solution (0.1 to 3 ml/g), NOC7 (an NO releasing agent, above 1 µg/kg), and a low dose of Alb-SNO (10 nmol/kg). However, blood pressure was not significantly reduced by NO solution or Alb-SNO. Intravenous injection of a high dose of Alb-SNO (300 nmol/kg) significantly reduced blood pressure with the appearance not only Alb-SNO in micromolar level in plasma, but also G-SNO in lesser degree. Conversely, the hypotensive effect of Cys-NO (300 nmol/kg, i.v.) and G-SNO (300 nmol/kg, i.v.) accompanied development of Alb-SNO (micromolar level), but not Cys-NO or G-SNO in plasma. R-SNOs were not found in plasma during profound hypotension induced by acetylcholine (10 and 30 µg/kg/min, continuous i.v.), glyceryl trinitrate (100 µg/kg, i.v.), sodium nitroprusside (100 µg/kg, i.v.), and isosorbide dinitrate (300 µg/kg, i.v.). These results indicate that R-SNOs do not play an important role under unstimulated condition. In addition, plasma R-SNOs may not be involved in pharmacological vasodilation where contributions of NO or R-SNOs are suggested.

Different disappearance rates of plasma nitrite (NO(2)(-)) contribute to apparent steady-state arterio-venous differences in anesthetized animals.

A possible cause of arterio-venous (A-V) differences in plasma nitrite (NO(2)(-)) levels under steady-state conditions and kinetic features of NO(2)(-) in arterial and venous blood were examined. In isolated rabbit blood, plasma NO(2)(-) in venous blood disappeared faster than that in arterial blood and was accompanied by a concomitant increase in nitrate (NO(3)(-)), implicating oxidation as the main pertinent metabolic pathway. When data were corrected with respective elimination constants and time durations before plasma separation, no A-V difference was estimated under steady-state. Even after these corrections for NO(2)(-) loading in anesthetized rabbits, a large A-V difference in NO(2)(-) levels (arterial venous) was observed, followed by an exponential decrease in NO(2)(-) levels without a reciprocal increase in NO(3)(-) levels. There was a marked difference in NO(2)(-) decay between in vivo and ex vivo experiments, but no increases in the circulating blood were detected for other substances derived from NO(2)(-), such as methemoglobin or low- and high-molecular weight nitrosothiols. In rats and guinea pigs, absence and presence of the A-V difference were detected under steady-state conditions and after NO(2)(-) loading, respectively. These observations indicate that apparent A-V differences under steady-state are artifacts arising from different rates of NO(2)(-) disappearance in arterial versus venous plasma during sample handling, and that tissue compartments may contribute to changes in NO(2)(-) levels in circulating blood. Therefore, caution is required when evaluating plasma NO(2)(-) levels, especially in venous blood.