Pharmacological and biochemical approaches were used to elucidate the involvement of growth factor signaling pathways mediating estrogen neuroprotection in primary cortical neurons after glutamate excitotoxicity. We addressed the activation of mitogen-activated protein kinase (MAPK) signaling pathways, which are activated by growth factors such as nerve growth factor (NGF). Inhibition of MAPK signaling with the MAPK kinase inhibitor PD98059 blocks both NGF and estrogen neuroprotection in these neurons. These results correlate with a rapid and sustained increase in MAPK activity within 30 min of estrogen exposure. The involvement of signaling molecules upstream from MAPK was also examined to determine whether activation of MAPK by estrogen is mediated by tyrosine kinase activity. Estrogen produces a rapid, transient activation of src-family tyrosine kinases and tyrosine phosphorylation of p21(ras)-guanine nucleotide activating protein. Effects of estrogen on neuroprotection, as well as rapid activation of tyrosine kinase and MAPK activity, are blocked by the anti-estrogen ICI 182,780. This provides evidence that activation of the MAPK pathway by estrogen participates in mediating neuroprotection via an estrogen receptor. These results describe a novel mechanism by which cytoplasmic actions of the estrogen receptor may activate the MAPK pathway, thus broadening the understanding of effects of estrogen in neurons.

No abstract

Recently, overexpression of the genes TMEM16A and TMEM16B has been shown to produce currents qualitatively similar to native Ca(2+)-activated Cl(-) currents (I(ClCa)) in vascular smooth muscle. However, there is no information about this new gene family in vascular smooth muscle, where Cl(-) channels are a major depolarizing mechanism. Qualitatively similar Cl(-) currents were evoked by a pipette solution containing 500 nM Ca(2+) in smooth muscle cells isolated from BALB/c mouse portal vein, thoracic aorta, and carotid artery. Quantitative PCR using SYBR Green chemistry and primers specific for transmembrane protein (TMEM) 16A or the closely related TMEM16B showed TMEM16A expression as follows: portal vein > thoracic aorta > carotid artery > brain. In addition, several alternatively spliced variant transcripts of TMEM16A were detected. In contrast, TMEM16B expression was very low in smooth muscle. Western blot analysis with different antibodies directed against TMEM16A revealed a number of products with a consistent band at ∼120 kDa, except portal vein, where an 80-kDa band predominated. TMEM16A protein was identified in the smooth muscle layers of 4-μm-thick slices of portal vein, thoracic aorta, and carotid artery. In isolated myocytes, fluorescence specific to a TMEM16A antibody was detected diffusely throughout the cytoplasm, as well as near the membrane. The same antibody used in Western blot analysis of lysates from vascular tissues also recognized an ∼147-kDa mouse TMEM16A-green fluorescent protein (GFP) fusion protein expressed in HEK 293 cells, which correlated to a similar band detected by a GFP antibody. Patch-clamp experiments revealed that I(ClCa) generated by transfection of TMEM16A-GFP in HEK 293 cells displayed remarkable similarities to I(ClCa) recorded in vascular myocytes, including slow kinetics, steep outward rectification, and a response similar to the pharmacological agent niflumic acid. This study shows that TMEM16A expression is robust in murine vascular smooth muscle cells, consolidating the view that this gene is a viable candidate for the native Ca(2+)-activated Cl(-) channel in this cell type.

N. Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension. Pulmonary artery smooth muscle cells (PASMCs) are more depolarized and display higher Ca 2ϩ levels in pulmonary hypertension (PH). Whether the functional properties and expression of Ca 2ϩ -activated ClϪ channels (Cl Ca), an important excitatory mechanism in PASMCs, are altered in PH is unknown. The potential role of Cl Ca channels in PH was investigated using the monocrotaline (MCT)-induced PH model in the rat. Three weeks postinjection with a single dose of MCT (50 mg/kg ip), the animals developed right ventricular hypertrophy (heart weight measurements) and changes in pulmonary arterial flow (pulse-waved Doppler imaging) that were consistent with increased pulmonary arterial pressure and PH. Whole cell patch experiments revealed an increase in niflumic acid (NFA)-sensitive Ca 2ϩ -activated Cl Ϫ current [ICl(Ca)] density in PASMCs from large conduit and small intralobar pulmonary arteries of MCT-treated rats vs. aged-matched saline-injected controls. Quantitative RT-PCR and Western blot analysis revealed that the alterations in I Cl(Ca) were accompanied by parallel changes in the expression of TMEM16A, a gene recently shown to encode for Cl Ca channels. The contraction to serotonin of conduit and intralobar pulmonary arteries from MCT-treated rats exhibited greater sensitivity to nifedipine (1 M), an L-type Ca 2ϩ channel blocker, and NFA (30 or 100 M, with or without 10 M indomethacin to inhibit cyclooxygenases) or T16A Inh-A01 (10 M), TMEM16A/Cl Ca channel inhibitors, than that of control animals. In conclusion, augmented Cl Ca/TMEM16A channel activity is a major contributor to the changes in electromechanical coupling of PA in this model of PH. TMEM16A-encoded channels may therefore represent a novel therapeutic target in this disease. pulmonary arterial tone; TMEM16A; anoctamin-1; Ca 2ϩ -activated Cl Ϫ channel; patch-clamp technique PULMONARY HYPERTENSION (PH) is defined as a sustained high blood pressure (Ͼ25 mmHg at rest and Ͼ30 mmHg during exercise) in the main pulmonary artery (PA) that ultimately leads to failure of the right hand side of the heart and death (4). Characteristic pathophysiological manifestations of PH are enhanced vasoconstriction, thickening of the arterial muscle wall, and a propensity for thrombosis, as a result of changes in all layers of the blood vessel, but little is known about the molecular mechanisms that drive these pathological responses. It is well established that pulmonary arterial smooth muscle cells (PASMCs) from animal models of PH and human PH patients are more depolarized and exhibit a higher intracellular calcium concentration ([Ca 2ϩ ] i ) than cells from healthy individuals and several ionic conductances are altered in PASMCs from animal models of PH and PH patients (4,13,29,43,68,70). Except for one recent study carried out using the chronic hypoxic model of PH in the rat (58), there is little information regarding the potential role of Ca 2ϩ -activa...

The signal transduction pathways regulating smooth-muscle gene expression and production of cytokines in response to proinflammatory mediators are undefined. Cultured human bronchial smooth-muscle cells were treated for 20 h with a cytokine cocktail containing interleukin (IL)-1beta, tumor necrosis factor-alpha, and interferon-gamma. A complementary DNA expression array containing 588 genes was used to follow cytokine-stimulated gene expression. The expression and secretion of the cytokines IL-1beta, IL-6, and IL-8 significantly increased after 20 h of stimulation as measured by relative reverse transcriptase/ polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting techniques. Expression of IL-6 and IL-8 was sensitive to SB203580, the specific inhibitor of p38 mitogen-activated protein (MAP) kinase and PD98059, an inhibitor of MAP kinase kinase. Expression of IL-1beta was sensitive only to PD98059. Together, these results demonstrate that the p38 and extracellular signal-regulated protein kinase MAP kinase pathways are required for proinflammatory mediator- induced cytokine expression in airway myocytes. The generation of chemokines and cytokines in airway smooth muscle also provides evidence that smooth-muscle cells have the ability to contribute to the inflammatory response.

Cognitive deficits associated with aging and with neurodegenerative diseases such as Alzheimer's disease have been attributed to degeneration of cholinergic neurons in the basal forebrain. Estrogen is known to provide trophic support to cholinergic neurons, although the mechanisms underlying the actions of estrogen have yet to be determined. Because cholinergic neurons require neurotrophic growth factors for their survival, it is possible that the trophic effects of estrogen on basal forebrain systems are caused by enhanced expression of neurotrophins or their receptors. To begin to examine this hypothesis, we used in situ hybridization analysis to determine the effects of ovariectomy (ovx) and estrogen replacement on trkA mRNA levels in the rat basal forebrain. Ten days of estrogen deprivation after ovx resulted in significant decreases in trkA mRNA levels in the horizontal limb of the diagonal band of Broca and the nucleus basalis of Meynert. Short-term estrogen replacement therapy restored trkA mRNA expression to a level comparable with ovary-intact animals. No changes in trkA mRNA levels were observed in the vertical limb of the diagonal band of Broca after ovx or estrogen replacement. To assess the functional status of cholinergic neurons in the absence and presence of estrogen, the effects of ovx and estrogen replacement on ChAT mRNA levels were also examined and found to reflect the changes observed in trkA mRNA expression. These studies suggest that the trophic effects of estrogen on basal forebrain cholinergic systems may be mediated, in part, through the signaling of neurotrophic growth factors through their receptors.

Previous studies in pulmonary arterial smooth muscle cells (PASMCs)

Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.