The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory ...responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing. Two of these variants (one lacking the first transmembrane domain, the second lacking the entire cytoplasmic tail) were compared to the full-length channel. Real-time PCR analysis demonstrated that both variants were expressed in various tissues and that the cytoplasmic tail deleted variant is highly expressed. Deletion of the first transmembrane domain resulted in a non-functional channel. Deletion of the cytoplasmic tail did not affect ion movement but severely affected the ability to form a large pore and to induce activation of caspases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial ...fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348±5451, which was blocked by spironolactone (0.3–60mg/kg, p.o.) and eplerenone (0.3–30mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10µM) and spironolactone (10µM) diminished lung hydroxyproline concentrations by 55±5, 122±9, and 83±8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10–60mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
3.
Optical sensing of biomolecules using microring resonators Yalcin, A.; Popat, K.C.; Aldridge, J.C. ...
IEEE journal of selected topics in quantum electronics,
2006-Jan.-Feb., 2006-01-00, 20060101, Volume:
12, Issue:
1
Journal Article
Peer reviewed
A biosensor application of vertically coupled glass microring resonators with Q/spl sim/12 000 is introduced. Using balanced photodetection, very high signal to noise ratios, and thus high ...sensitivity to refractive index changes (limit of detection of 1.8/spl times/10/sup -5/ refractive index units), are achieved. Ellipsometry and X-ray photoelectron spectroscopy results indicate successful modification of biosensor surfaces. Experimental data obtained separately for a bulk change of refractive index of the medium and for avidin-biotin binding on the ring surface are reported. Excellent repeatability and close-to-complete surface regeneration after binding are experimentally demonstrated.
The Transient Receptor Potential cation channel V1 (TRPV1) is expressed in peripheral nociceptive neurons and is subject to polymodal activation via various agents including capsaicin, noxious heat, ...low extracellular pH, and direct phosphorylation by protein kinase C (PKC). We have cloned and heterologously expressed mouse TRPV1 (mTRPV1) and characterized its function utilizing FLIPR-based calcium imaging to measure functional responses to various small molecule agonists, low pH and direct phosphorylation via PKC. The various TRPV1 agonists activated mTRPV1 with a rank order of agonist potency of (resiniferatoxin (RTX)
=
arvanil
>
capsaicin
=
olvanil
>
OLDA
>
PPAHV) (EC
50 values of 0.15
±
0.04
nM, 0.27
±
0.07
nM, 9.1
±
1.2
nM, 3.7
±
0.3
nM, 258
±
105
nM, and 667
±
151
nM, respectively). Additionally, mTRPV1 was activated by either low pH or with addition of the PKC activator phorbol 12-myristate 13-acetate (PMA). The TRPV1 antagonists iodinated-resiniferatoxin (I-RTX) or BCTC were both able to block capsaicin, pH and PKC-induced responses of mTRPV1 (IC
50 (I-RTX)
=
0.35
±
0.12
nM, 1.9
±
0.7
nM, and 0.80
±
0.68
nM, IC
50 (BCTC)
=
1.3
±
0.36
nM, 0.59
±
0.16
nM, and 0.37
±
0.15
nM, respectively). However, the antagonist capsazepine was only able to inhibit a capsaicin-evoked response of mTRPV1 with an IC
50 of 1426
±
316
nM. Comparable results were achieved with rat TRPV1, while capsazepine blocked all modes of human TRPV1 activation. Thus, the mTRPV1 cation channel has a molecular pharmacological profile more akin to rat TRPV1 than either human or guinea pig TRPV1 and the molecular pharmacology suggests that capsazepine may be an ineffective TRPV1 antagonist for in vivo models of inflammatory pain in the mouse.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Histamine exerts its numerous physiological functions through interaction with G protein-coupled receptors. Three such receptors have been defined at both the pharmacological and molecular level, ...while pharmacological evidence hints at the existence of further subtypes. We report here the cloning and characterization of a fourth histamine receptor subtype. Initially discovered in an expressed-sequence tag database, the full coding sequence (SP9144) was subsequently identified in chromosome 18 genomic sequence. This virtual coding sequence exhibited highest homology to the H(3) histamine receptor and was used to generate a full-length clone by polymerase chain reaction (PCR). The distribution of mRNA encoding SP9144 was restricted to cells of the immune system as determined by quantitative PCR. HEK-293 cells transiently transfected with SP9144 and a chimeric G protein alpha-subunit (Galpha(q/i1,2)) exhibited increases in intracellular Ca(2+) in response to histamine but not other biogenic amines. SP9144-transfected cells exhibited saturable, specific, high-affinity binding of (3)Hhistamine, which was potently inhibited by H(3) receptor-selective compounds. The rank order and potency of these compounds at SP9144 differed from the rank order at the H(3) receptor. Although SP9144 apparently coupled to Galpha(i), HEK-293 cells stably transfected with SP9144 did not exhibit histamine-mediated inhibition of forskolin-stimulated cAMP levels. However, both (35)SGTPgammaS binding and phosphorylation of mitogen-activated protein kinase were stimulated by histamine via SP9144 activation. In both of these assays, SP9144 exhibited evidence of constitutive activation. Taken together, these data demonstrate that SP9144 is a unique, fourth histamine receptor subtype.
SCH351591, a novel phosphodiesterase-4 inhibitor under investigation as a potential therapeutic for asthma and chronic obstructive pulmonary disease (COPD), was evaluated in a 3-month rising-dose ...study in Cynomolgus monkeys. Four groups, containing four monkeys/sex, received vehicle control or rising doses up to 12, 24, or 48 mg/kg of SCH351591 daily. Although initial exposure produced clinical signs of emesis, reduced food intake, and reduced body weight, tachyphylaxis to the emesis allowed dose escalation up to 48 mg/kg/day. Two monkeys died and 3 were sacrificed in moribund condition over the course of the study. Early mortality, involving monkeys dosed with 12 or 24 mg/kg, was attributed to sepsis (2 monkeys) or colon inflammation (3 monkeys). Leukocyte function assays on low- and mid-dose group survivors revealed an inhibition of T lymphocyte proliferation for 12 mg/kg group males and 24 mg/kg group monkeys of both sexes. Necropsy findings, unassociated with early mortality, included reduced size and weight of the thymus, depletion of body fat, red discoloration of the gastric mucosa, and perivascular hemorrhage of the stomach and heart. Stomach and heart gross findings were present in the high-dose group only. Histopathologic lesions, in addition to those attributed to concurrent bacterial infection, included thymic atrophy, serous atrophy of fat, myocardial degeneration and acute to chronic inflammation of small to medium-sized arteries in various organs and tissues including the heart, kidneys, stomach, salivary glands, pancreas, esophagus, gallbladder, and mesentery. The findings of this study demonstrate the potential of a PDE4 inhibitor to alter immunologic response as well as to produce arteriopathy in nonhuman primates.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific cation channel capable of integrating various noxious chemical and physical stimuli. The dog orthologue of TRPV1 ...was cloned using cDNA from nodose ganglia and heterologously expressed in HEK293
OFF cells. At the amino acid level, dTRPV1 displays 85–89% sequence identity to other TRPV1 orthologues. Molecular pharmacological characterization of HEK293
OFF cells expressing TRPV1 was assessed using a fluorescence imaging plate reader (FLIPR)-based calcium imaging assay. Dog TRPV1 was activated by various known TRPV1 agonists in a concentration-dependent manner: Ag23
=
resiniferatoxin
>
olvanil
∼
arvanil
>
capsaicin
>
phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV)
>
N-oleoyldopamine (OLDA). In addition, select TRPV1 antagonists (capsazepine, I-resiniferatoxin and
N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2
H)-carbox-amide (BCTC)) were able to block the response of dTRPV1 to capsaicin. Furthermore, the dog TRPV1 lacked a conserved protein kinase A (PKA) phosphorylation site (117) found in other cloned orthologues, which may have physiological consequences on dog TRPV1 function
. Taken together, these data constitute the first study of the cloning, expression and pharmacological characterization of dog TRPV1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cough is an important defensive pulmonary reflex that removes irritants, fluids, or foreign materials from the airways. However, when cough is exceptionally intense or when it is chronic and/or ...nonproductive it may require pharmacologic suppression. For many patients, antitussive therapies consist of OTC products with inconsequential efficacies. On the other hand, the prescription antitussive market is dominated by older opioid drugs such as codeine. Unfortunately, “codeine-like” drugs suppress cough at equivalent doses that also often produce significant ancillary liabilities such as GI constipation, sedation, and respiratory depression. Thus, the discovery of a novel and effective antitussive drug with an improved side effect profile relative to codeine would fulfill an unmet clinical need in the treatment of cough. Afferent pulmonary nerves are endowed with a multitude of potential receptor targets, including TRPV1, that could act to attenuate cough. The evidence linking TRPV1 to cough is convincing. TRPV1 receptors are found on sensory respiratory nerves that are important in the generation of the cough reflex. Isolated pulmonary vagal afferent nerves are responsive to TRPV1 stimulation.
In vivo
, TRPV1 agonists such as capsaicin elicit cough when aerosolized and delivered to the lungs. Pertinent to the debate on the potential use of TRPV1 antagonist as antitussive agents are the observations that airway afferent nerves become hypersensitive in diseased and inflamed lungs. For example, the sensitivity of capsaicin-induced cough responses following upper respiratory tract infection and in airway inflammatory diseases such as asthma and COPD is increased relative to that of control responses. Indeed, we have demonstrated that TRPV1 antagonism can attenuate antigen-induced cough in the allergic guinea pig. However, it remains to be determined if the emerging pharmacologic profile of TRPV1 antagonists will translate into a novel human antitussive drug. Current efforts in clinical validation of TRPV1 antagonists revolve around various pain indications; therefore, clinical evaluation of TRPV1 antagonists as antitussive agents will have to await those outcomes.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The molecular and pharmacological properties of adenosine receptors in the T24 human bladder epithelial carcinoma cell line were assessed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), ...Ca
2+ Flux, cAMP production and interleukin-8 measurements. RT-PCR experiments detected the presence of transcripts for the adenosine A
1, A
2A and A
2B receptors but not for the adenosine A
3 subtype. Application of specific adenosine receptor ligands resulted in concentration-dependent increases in intracellular calcium (Ca
2+
i) with the following order of potency and EC
50 values: 5′-
N-Ethylcarboxamidoadenosine (NECA) (1153
±
214)
>
5′-(
N-Cyclopropyl)carboxamidoadenosine (CPCA) (1436
±
186)
>
adenosine (4823
±
932). This rank order of potency is typical of adenosine A
2B receptors. In addition, select adenosine receptor antagonists
N-(4-acetylphenyl)-2-4-(2,3,6,7-tetrahydro-2,6 dioxo-1,3-dipropyl-1
H-purin-8-yl)phenoxyacetamide (MRS 1706), 8-4-((4-Cyano2,6--phenyl)carbamoylmethyl)oxyphenyl-1,3-di(
n-propyl)-xanthine (MRS 1754), 1,3-Diethyl-8-phenylxanthine (DPCPX), 1,3-Diethyl-8-phenylxanthine (DPX), Alloxazine, 8-(3-Chlorostyryl)caffeine (CSC), and Theophylline blocked the NECA-induced calcium responses. Additionally, NECA, CPCA, and adenosine stimulated cAMP formation with a rank order of potency characteristic of adenosine A
2B receptors. The select adenosine A
2A antagonist, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo4,3-e-1,2,4-triazolo1,5-c pyrimidine (SCH 58261) failed to antagonize the NECA response, whereas the potent and highly selective adenosine A
2B antagonists MRS 1754 and MRS 1706 inhibited NECA-stimulated cAMP production. Lastly, NECA-induced interleukin-8 secretion was inhibited by MRS 1754. Taken together, these data indicate that Ca
2+
i accumulation and cAMP production as well as interleukin-8 secretion is mediated through the adenosine A
2B receptor in the T24 cell line.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
