TMPRSS3 encodes a transmembrane serine protease that contains both LDLRA and SRCR domains and is mutated in non-syndromic autosomal recessive deafness (DFNB8/10). To study its function, we cloned the mouse ortholog which maps to Mmu17, which is structurally similar to the human gene and encodes a polypeptide with 88% identity to the human protein. RT-PCR and RNA in situ hybridization on rat and mouse cochlea revealed that Tmprss3 is expressed in the spiral ganglion, the cells supporting the organ of Corti and the stria vascularis. RT-PCR on mouse tissues showed expression in the thymus, stomach, testis and E19 embryos. Transient expression of wild-type or tagged TMPRSS3 protein showed a primary localization in the endoplasmic reticulum. The epithelial amiloride-sensitive sodium channel (ENaC), which is expressed in many sodium-reabsorbing tissues including the inner ear and is regulated by membrane-bound channel activating serine proteases (CAPs), is a potential substrate of TMPRSS3. In the Xenopus oocyte expression system, proteolytic processing of TMPRSS3 was associated with increased ENaC mediated currents. In contrast, 6 TMPRSS3 mutants (D103G, R109W, C194F, W251C, P404L, C407R) causing deafness and a mutant in the catalytic triad of TMPRSS3 (S401A), failed to undergo proteolytic cleavage and activate ENaC. These data indicate that important signaling pathways in the inner ear are controlled by proteolytic cleavage and suggest: (i) the existence of an auto-catalytic processing by which TMPRSS3 would become active, and (ii) that ENaC could be a substrate of TMPRSS3 in the inner ear.

Cerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provide evidence in mice that this gender difference only occurs when the ischemic brain is reperfused. The limited tissue salvage achieved by reperfusion in male mice is associated with increased expression of proinflammatory proteins, including cyclooxygenase-2 (Cox-2), Nox2, and vascular cell adhesion molecule-1 (VCAM-1), and infiltration of Nox2-containing T lymphocytes into the infarcted brain, whereas such changes are minimal in female mice after ischemia-reperfusion (I-R). Infarct volume after I-R was no greater at 72 h than at 24 h in either gender. Infarct development was Nox2 dependent in male but not in female mice, and Nox2 within the infarct was predominantly localized in T lymphocytes. Stroke resulted in an B15-fold increase in Nox2-dependent superoxide production by circulating, but not spleen-derived, T lymphocytes in male mice, and this was Bsevenfold greater than in female mice. These circulating immune cells may thus represent a major and previously unrecognized source of superoxide in the acutely ischemic and reperfused brain of males (and potentially in postmenopausal females). Our findings provide novel insights into mechanisms that could be therapeutically targeted in acute ischemic stroke patients who receive thrombolysis therapy to induce cerebral reperfusion.

We have previously shown that glucocorticoids inhibit mitogen-stimulated proliferation of human cultured airway smooth muscle (ASM) cells. The present study analyzed the effect of glucocorticoids on key regulatory pathways leading to passage of cells through the restriction point of the cell cycle, including those mediated by extracellular-regulated kinases (ERK) 1 and 2; the ERK upstream regulator MAPK kinase (MEK1); cyclin D1 levels; and levels and phosphorylation of retinoblastoma protein (pRb). Fluticasone propionate, a new inhaled glucocorticoid, was at least 10-fold more potent than dexamethasone in inhibiting thrombin-stimulated DNA synthesis and increases in cell number. Thrombin-stimulated increases in the levels and hyperphosphorylation of pRb were inhibited by glucocorticoids, which also reduced thrombin-stimulated cyclin D1 protein and messenger RNA (mRNA) levels. PD98059 (10 microM), an inhibitor of MEK1 activation, markedly attenuated thrombin stimulation of ERK activity and phosphorylation, DNA synthesis, and cyclin D1 levels. However, glucocorticoids had no effect on ERK activity or phosphorylation at 5 min, 2 h, or 12 h after addition of thrombin. In conclusion, glucocorticoid-induced reduction of cyclin D1 mRNA and protein levels, and of pRb phosphorylation, is sufficient to account for inhibition of ASM proliferation. Furthermore, these inhibitory effects of glucocorticoids on cyclin D1 and pRb occur on a component of the mitogen signaling cascade that is either downstream of or parallel to the ERK pathway.

M acrophages accumulate in the vascular wall during hypertension and likely contribute to the oxidative stress, endothelial dysfunction, and vascular inflammation that are hallmarks of the condition and that ultimately contribute to clinically relevant end points, such as atherosclerosis and arterial remodeling and stiffening.1,2 The circulating precursors of macrophages are monocytes, and depletion of these cells in mice is known to provide protection against experimentally induced hypertension. 3 Chemokines are chemotactic cytokines that can be released from cells at sites of injury or infection. By binding to specific receptors expressed on the surface of leukocytes, chemokines stimulate the extravasation and accumulation of leukocytes at sites of damage. Receptors for chemokines belong to the G-protein-coupled receptor superfamily, the largest and most tractable drug targets in the human genome.4-6 Twenty chemokine receptors have been identified to date, each of which may be stimulated by one or several chemokine ligands. 5In the present study, we performed a polymerase chain reaction (PCR) screen to identify chemokine receptor genes that are upregulated in the vascular wall of mice after induction of hypertension by treatment with a combination of deoxycorticosterone acetate (DOCA) and salt. Having identified CCR2 as one such chemokine receptor, we then examined the effect of a recently described and highly selective CCR2 antagonist, INCB3344, 7-9 on macrophage accumulation and blood pressure (BP) in the DOCA/salt model. Treatment of mice with INCB3344 reversed DOCA/ salt-induced increases in CCR2 expression and macrophage accumulation in the vascular wall. Importantly, these effects were accompanied by a reduction in BP, highlighting CCR2 as a promising drug target in hypertension.Abstract-Infiltration of macrophages into the artery wall plays detrimental roles during hypertension by promoting vascular inflammation and endothelial dysfunction, and it occurs via a chemo-attractant action of chemokines on macrophage cytokine receptors. We sought to identify the key chemokine receptors associated with macrophage infiltration into the vascular wall during deoxycorticosterone acetate (DOCA)/salt-induced hypertension in mice and to evaluate the impact of pharmacological inhibition of these receptors on blood pressure and leukocyte accumulation. 10,11 Mice were anesthetized via intraperitoneal injection of ketamine (100 mg/kg; Parnell Laboratories, Australia) and xylazine (10 mg/kg; Troy Laboratories), and a dorsal-lateral incision was made through the skin and muscle layers to expose the left kidney. The renal artery was then tied off with sutures and cut, distal to the ligature, to allow removal of the kidney. Before closing, the same incision site was used to implant a 21-day continuous-release DOCA pellet (2.4 mg/d; Innovative Research of America) subcutaneously in the scapular region. Finally, the drinking water was replaced with 0.9% saline. In sham-treated animals, the kidney was exposed but not removed, a...

Recent years have witnessed the discovery of novel selective agonists of the M(1) muscarinic acetylcholine (ACh) receptor (mAChR). One mechanism invoked to account for the selectivity of such agents is that they interact with allosteric sites. We investigated the molecular pharmacology of two such agonists, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) and 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), at the wild-type M(1) mAChR and three mutant M(1) mAChRs. Both agonists inhibited the binding of the orthosteric antagonist [(3)H]N-methyl scopolamine ([(3)H]NMS) in a manner consistent with orthosteric competition or high negative cooperativity. Functional interaction studies between 77-LH-28-1 and ACh also indicated a competitive mechanism. Dissociation kinetics assays revealed that the agonists could bind allosterically when the orthosteric site was prelabeled with [(3)H]NMS and that 77-LH-28-1 competed with the prototypical allosteric modulator heptane-1,7-bis-[dimethyl-3'-phthalimidopropyl]-ammonium bromide under these conditions. Mutation of the key orthosteric site residues Y(381)A (transmembrane helix 6) and W(101)A (transmembrane helix 3) reduced the affinity of prototypical orthosteric agonists but increased the affinity of the novel agonists. Divergent effects were also noted on agonist signaling efficacies at these mutants. We identified a novel mutation, F(77)I (transmembrane helix 2), which selectively reduced the efficacy of the novel agonists in mediating intracellular Ca(2+) elevation and phosphorylation of extracellular signal regulated kinase 1/2. Molecular modeling suggested a possible "bitopic" binding mode, whereby the agonists extend down into the orthosteric site as well as up toward extracellular receptor regions associated with an allosteric site. It is possible that this bitopic mode may explain the pharmacology of other selective mAChR agonists.

O ur knowledge of estrogen signaling pathways during cerebral ischemia, including the relative importance of different estrogen receptors and identities of estrogen-regulated gene networks affecting neuronal death, remains incomplete. Until recently, estrogen was thought to elicit its neuroprotective actions solely via activation of the classical nuclear receptors, estrogen receptor(ER)α and ERβ.1,2 However, the discovery of a novel estrogen receptor called G protein-coupled estrogen receptor (GPER, formerly known as GPR30), with high expression in the brain 3,4 and cerebral circulation, 5 presents a third receptor that may influence estrogenmediated outcomes after stroke. Important differences exist between GPER and classical estrogen receptors in that GPER responds to estrogen with rapid cellular signaling, and it is a G protein-coupled receptor localized on the plasma membrane 6 and intracellular membranes such as the endoplasmic reticulum and Golgi apparatus. Similar to classical estrogen receptor signaling, there is evidence for neuroprotection after GPER activation in ovariectomized (OVX) animals subjected to stroke. Chronic pretreatment of OVX rodents with the selective GPER agonist, G-1, reduces brain injury after focal or global ischemia. 8,9Interestingly, we have found that GPER distribution and expression is increased in the brain of male mice, but not of intact female or OVX mice, after transient focal ischemia. 4This sex-dependent regulation of GPER expression after cerebral ischemia could help explain some of the complex effects of estrogen in the brain after stroke and also guide the Background and Purpose-Experimental studies indicate that estrogen typically, but not universally, has a neuroprotective effect in stroke. Ischemic stroke increases membrane-bound G protein-coupled estrogen receptor (GPER) distribution and expression in the brain of male but not female mice. We hypothesized that GPER activation may have a greater neuroprotective effect in males than in females after stroke. Methods-Vehicle (dimethyl sulfoxide), a GPER agonist (G-1, 30 μg/kg), or a GPER antagonist (G-15, 300 μg/kg) were administered alone or in combination to young or aged male mice, or young intact or ovariectomized female mice, 1 hour before or 3 hours after cerebral ischemia-reperfusion. Some mice were treated with a combination of G-1 and the pancaspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-V D -OPh), 1 hour before stroke. We evaluated functional and histological end points of stroke outcome up to 72 hours after ischemia-reperfusion. In addition, apoptosis was examined using cleaved caspase-3 immunohistochemistry. Results-Surprisingly, G-1 worsened functional outcomes and increased infarct volume in males poststroke, in association with an increased expression of cleaved caspase-3 in peri-infarct neurons. These effects were blocked by G-15 or Q-VDOPh. Conversely, G-15 improved functional outcomes and reduced infarct volume after stroke in males, whether given before or after stroke. In contrast to find...

We have investigated the glycosylation, disulfide bonding, and subunit structure of mouse TRPM8. To do this, aminoterminal c-myc or hemagglutinin epitope-tagged proteins were incorporated and expressed in Chinese hamster ovary cells. These modifications had no obvious effects on channel function in intracellular calcium imaging assays upon application of agonists, icilin or menthol, and cold temperatures. Unmodified TRPM8 migrates with an apparent mass of 129 kDa and can be glycosylated in Chinese hamster ovary cells to give glycoproteins with apparent masses of 136 and 147 kDa. We identified two potential N-linked glycosylation sites in TRPM8 (Asn-821 and Asn-934) and mutated them to show that only the site in the putative pore region at position 934 is modified and that glycosylation of this site is not absolutely necessary for cell surface expression or responsiveness to icilin, menthol, and cool temperatures. Enzymatic cleavage of the carbohydrate chains indicated that they are complex carbohydrate. The glycosylation site is flanked in the pore by two cysteine residues that we mutated, to prove that they are involved in a conserved double cysteine motif, which is essential for channel function. Mutation of either of these cysteines abolishes function and forces the formation of a non-functional complex of the size of a homodimer. The double cysteine mutant is also non-functional. Finally, we showed in Perfluoro-octanoic acid-polyacrylamide gels that TRPM8 can form a tetramer (in addition to dimer and trimer forms), consistent with current thinking that functional TRP ion channels are tetrameric.In mammals, temperature is sensed through primary afferent sensory neurons whose cell bodies are located in the dorsal root and trigeminal ganglia. Signals from these cells are transmitted to the spinal cord and then to the brain where they are integrated and deciphered to evoke reflexive and cognitive responses. Growing evidence suggests that the principal molecular thermosensors in the sensory neurons belong to the family of transient receptor potential (TRP) 2 channels. Six temperature-sensitive TRP channels have been identified so far, four belonging to the TRPV subfamily and sensing heat (TRPV1, TRPV2, TRPV3, and TRPV4) and TRPM8 and TRPA1, which are sensitive to cold temperatures. Some of these channels are also sensitive to compounds that mimic the sensation of heat or cold, for example, TRPV1 responds to capsaicin, the pungent ingredient of "hot" chili pepper, and TRPM8 is sensitive to the cooling compounds menthol and icilin.The properties of TRPV1 have been extensively studied by electrophysiological, pharmacological, and biochemical methods (1). TRPM8 was cloned more recently (2, 3) and is less well studied.TRPM8 is a non-selective cation channel predominantly expressed in a subpopulation of thermoceptive/nociceptive neurons found in dorsal root ganglia and in trigeminal ganglia. In dorsal root ganglia, TRPM8 is found in 5-10% of neurons that have a small diameter and do not stain with antibodies to neuro...

1 The relationship between persistent ERK (extracellular signal-regulated kinase) activity, cyclin D1 protein and mRNA levels and cell cycle progression in human cultured airway smooth muscle was examined in response to stimulation by ET-1 (endothelin-1), thrombin and bFGF (basic ®broblast growth factor). 2 Thrombin (0.3 and 3 u ml 71 ) and bFGF (0.3 and 3 nM) increased ERK activity for more than 2 h and increased cell number, whereas ET-1 (100 nM) transiently stimulated ERK activity and was non-mitogenic. 3 The MEK1 (mitogen-activated ERK kinase) inhibitor, PD 98059 (30 mM), inhibited both ERK phosphorylation and activity, and either prevented (thrombin 0.3 and 3 u ml 71, bFGF 300 pM) or attenuated (bFGF 3 nM) DNA synthesis. 4 Thrombin and bFGF increased both cyclin D1 mRNA and protein levels. PD 98059 decreased cyclin D1 protein levels stimulated by the lower but not higher thrombin concentrations. Moreover, increases in cyclin D1 mRNA levels were unaected by PD 98059 pretreatment, irrespective of the mitogen or its concentration, suggesting that inhibition of cyclin D1 protein levels occurred by a post-transcriptional mechanism. 5 These ®ndings indicate that the control of cyclin D1 protein levels may occur independently of the MEK1/ERK signalling pathways. The inhibition of S phase entry by PD 98059 at higher thrombin concentrations appears to result from eects on pathways downstream or parallel to those regulating cyclin D1 protein levels. These ®ndings suggest heterogeneity in the signalling of DNA synthesis in human cultured airway smooth muscle.