Marilee J. Wick

Sites of alcohol and volatile anaesthetic action on GABA A and glycine receptors

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as γ-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABAA and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA ρ1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABAA and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.

Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) Expression Is Decreased in Pulmonary Hypertension and Affects Endothelial Cell Growth

Abstract— PPAR&ggr; is a member of a family of nuclear receptors/ligand–dependent transcription factors, which bind to hormone response elements on target gene promoters. An antiproliferative and proapoptotic action profile of PPAR&ggr; has been described and PPAR&ggr; may function as a tumor suppressor gene, but little is known about the role of PPAR&ggr; in vascular remodeling. One group of human diseases that shows impressive vascular remodeling exclusively in the lungs is the group of severe pulmonary hypertensive disorders, which is characterized by complex, endothelial cell–proliferative lesions of lung precapillary arterioles composed of clusters of phenotypically altered endothelial cells that occlude the vessel lumen and contribute to the elevation of the pulmonary arterial pressure and reduce local lung tissue blood flow. In the present study, we report the ubiquitous PPAR&ggr; expression in normal lungs, and in contrast, a reduced lung tissue PPAR&ggr; gene and protein expression in the lungs from patients with severe PH and loss of PPAR&ggr; expression in their complex vascular lesions. We show that fluid shear stress reduces PPAR&ggr; expression in ECV304 endothelial cells, that ECV304 cells that stably express dominant-negative PPAR&ggr; (DN-PPAR&ggr; ECV304) form sprouts when placed in matrigel and that DN-PPAR&ggr; ECV304 cells, after tail vein injection in nude mice, form lumen-obliterating lung vascular lesions. We conclude that fluid shear stress decreases the expression of PPAR&ggr; in endothelial cells and that loss of PPAR&ggr; expression characterizes an abnormal, proliferating, apoptosis-resistant endothelial cell phenotype.

Neprilysin Null Mice Develop Exaggerated Pulmonary Vascular Remodeling in Response to Chronic Hypoxia

Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder.

Rescue of γ2 subunit‐deficient mice by transgenic overexpression of the GABAA receptor γ2S or γ2L subunit isoforms

The γ2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high‐affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the γ2 subunit, γ2 short (γ2S) and γ2 long (γ2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the γ2S or γ2L subunit on a γ2 subunit‐deficient background are phenotypically indistinguishable from wild‐type. They express nearly normal levels of γ2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild‐type in both juvenile and adult mice. Our results indicate that the two γ2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform‐specific roles in phosphorylation‐dependent regulatory mechanisms.

Decreased Neprilysin and Pulmonary Vascular Remodeling in Chronic Obstructive Pulmonary Disease

RATIONALE Studies with genetically engineered mice showed that decreased expression of the transmembrane peptidase neprilysin (NEP) increases susceptibility to hypoxic pulmonary vascular remodeling and hypertension; in hypoxic wild-type mice, expression is decreased early in distal pulmonary arteries, where prominent vascular remodeling occurs. Therefore, in humans with smoke- and hypoxia-induced vascular remodeling, as in chronic obstructive pulmonary disease (COPD), pulmonary activity/expression of NEP may likewise be decreased. OBJECTIVES To test whether NEP activity and expression are reduced in COPD lungs and pulmonary arterial smooth muscle cells (SMCs) exposed to cigarette smoke extract or hypoxia and begin to investigate mechanisms involved. METHODS Control and advanced COPD lung lysates (n = 13-14) were analyzed for NEP activity and protein and mRNA expression. As a control, dipeptidyl peptidase IV activity was analyzed. Lung sections were assessed for vascular remodeling and oxidant damage. Human pulmonary arterial SMCs were exposed to cigarette smoke extract, hypoxia, or H₂O₂, and incubated with antioxidants or lysosomal/proteasomal inhibitors. MEASUREMENTS AND MAIN RESULTS COPD lungs demonstrated areas of vascular rarification, distal muscularization, and variable intimal and prominent medial/adventitial thickening. NEP activity was reduced by 76%; NEP protein expression was decreased in alveolar walls and distal vessels; mRNA expression was also decreased. In SMCs exposed to cigarette smoke extract, hypoxia, and H₂O₂, NEP activity and expression were also reduced. Reactive oxygen species inactivated NEP activity; NEP protein degradation appeared to be substantially induced. CONCLUSIONS Mechanisms responsible for reduced NEP activity and protein expression include oxidative reactions and protein degradation. Maintaining or increasing lung NEP may protect against pulmonary vascular remodeling in response to chronic smoke and hypoxia.

An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

Protection against vascular leak in neprilysin transgenic mice with complex overexpression pattern

Neprilysin (NEP) is a cell surface metallopeptidase found in many tissues. Based mostly on pharmacological manipulations, NEP has been thought to protect blood vessels from plasma extravasation. We have suggested that NEP may protect against pulmonary vascular injury. However, these prior studies did not utilize mice which overexpress NEP. The aims of the present investigation were to develop and characterize doubly transgenic (DT) mice that overexpress NEP universally and conditionally, and to investigate the protective effect that overexpressed NEP may have against plasma extravasation in the vasculature. The duodenum, which is often used to assess vascular permeability, and in which the NEP protein was overexpressed in our DT mice two-fold, was selected as our experimental preparation. We found that substance P-induced plasma extravasation was decreased substantially (3.5-fold) in the duodenums of our doxycycline-treated DT mice, giving independent evidence of NEP’s protective effects against plasma extravasation. Transgenic lung NEP protein was not stably expressed in the DT mice, so we were not able to test the effect of NEP overexpression in the lung. Although initially overexpressed nearly nine-fold at that site, pulmonary NEP protein overexpression eventually dissipated. Surprisingly, at a time when there was no lung transgenic NEP protein overexpression, lung NEP mRNA expression was still increased 23-fold, indicating that the expression defect probably is not transcriptional. These studies help to characterize our complex transgenic model of NEP overexpression and further demonstrate NEP’s protective effects against plasma extravasation.