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 gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 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 GABA(A) 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.

Alcohols in the homologous series of nalcohols increase in central nervous system depressant potency with increasing chain length until a ''cutoff'' is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the ␣ subunit. We now demonstrate that these residues in the glycine ␣1 and the ␥-aminobutyric acid 1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine ␣1 receptor, whereas mutation of Ile-307 and͞or Trp-328 in the ␥-aminobutyric acid 1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

PPARgamma 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 PPARgamma has been described and PPARgamma may function as a tumor suppressor gene, but little is known about the role of PPARgamma 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 PPARgamma expression in normal lungs, and in contrast, a reduced lung tissue PPARgamma gene and protein expression in the lungs from patients with severe PH and loss of PPARgamma expression in their complex vascular lesions. We show that fluid shear stress reduces PPARgamma expression in ECV304 endothelial cells, that ECV304 cells that stably express dominant-negative PPARgamma (DN-PPARgamma ECV304) form sprouts when placed in matrigel and that DN-PPARgamma 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 PPARgamma in endothelial cells and that loss of PPARgamma expression characterizes an abnormal, proliferating, apoptosis-resistant endothelial cell phenotype.

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Glycine and ␥-aminobutyric acid (GABA) A receptors are members of the "superfamily" of ion channels, and are sensitive to allosteric modulation by n-alcohols such as ethanol and butanol. We recently demonstrated that the mutation of Ser-267 to Ile in the ␣1 subunit abolished ethanol regulation of glycine receptors (Gly-R). In the present study, a pair of chimeric receptors was studied, in which a 45-amino acid domain comprising transmembrane domains 2 and 3 was exchanged between the Gly-R␣1 and ␥-aminobutyric acid 1 subunits. Detailed pharmacologic analysis of these chimeras confirmed that this domain of the Gly-R confers enhancement of receptor function by ethanol and butanol. An extensive series of mutations at Ser-267 in the Gly-R␣1 subunit was also prepared, and the resulting homomeric receptors were expressed and tested for sensitivity to glycine, and allosteric modulation by alcohols. All of the mutant receptors expressed successfully in Xenopus oocytes. Mutation of Ser-267 to small amino acid residues such as Gly or Ala produced receptors in which glycine responses were potentiated by ethanol. As we have reported previously, the mutant Gly-R␣1 (Ser-267 3 Ile) was completely insensitive to ethanol; mutation of Ser-267 to Val had a similar effect. Mutation of Ser-267 to large residues such as His, Cys, or Tyr resulted in inhibition of Gly-R function by ethanol. These results demonstrate that the size of the amino acid residue at position ␣267 plays a crucial role in determining the functional consequences of allosteric modulation of the Gly-R by alcohols.Depression of central nervous system function by ethanol is a complex phenomenon (1, 2), which may involve several neurotransmitter systems. Ligand-gated ion channels play an important role in the regulation of neuronal excitability and are sensitive to pharmacologically relevant concentrations of ethanol. Several such channels are thought to be involved in the behavioral effects of ethanol, for example the NMDA 1 and non-NMDA subtypes of glutamate receptors (3-5) and the GABA A receptors (for review, see Ref. 6). Recent data also suggest that the related glycine receptor (Gly-R) may also be an important site of alcohol action. Gly-Rs constitute the major inhibitory neurotransmitter receptor system in the brainstem and spinal cord but are also found in significant numbers in higher brain regions such as the olfactory bulb, midbrain, cerebellum, and cerebral cortex (7). Evidence has recently been obtained to implicate the Gly-R in some of the behavioral effects of ethanol. Intracerebroventricular administration of glycine augments ethanol-induced loss of righting reflex in the mouse (8), and this effect is blocked by the glycine antagonist strychnine. Enhancement of Gly-R function would be consistent with the behavioral actions of ethanol in vivo. Electrophysiological studies have shown that ethanol enhances Gly-R function in spinal cord neurons from mouse and chick (9, 10), and increases glycine-mediated Cl Ϫ uptake into rat brain synaptoneurosomes (11). Re...

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One enigma in tumor immunology is why animals bearing malignant grafts can reject normal grafts that express the same nonself-antigen. An explanation for this phenomenon could be that different T cell clones react to the normal graft and the malignant cells, respectively, and only the tumor-reactive clonotypes may be affected by the growing tumor. To test this hypothesis, we used a T cell receptor transgenic mouse in which essentially all CD8+ T cells are specific for a closely related set of self-peptides presented on the MHC class I molecule Ld. We find that the tumor expressed Ld in the T cell receptor transgenic mice but grew, while the Ld-positive skin was rejected. Thus, despite an abundance of antigen-specific T cells, the malignant tissue grew while normal tissue expressing the same epitopes was rejected. Therefore, systemic T cell exhaustion or anergy was not responsible for the growth of the antigenic cancer cells. Expression of costimulatory molecules on the tumor cells after transfection and preimmunization by full-thickness skin grafts was required for rejection of a subsequent tumor challenge, but there was no detectable effect of active immunization once the tumor was established. Thus, the failure of established tumors to attract and activate tumor-specific T cells at the tumor site may be a major obstacle for preventive or therapeutic vaccination against antigenic cancer.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the growth of different cancer cell types, suggesting a broad role for their cyclooxygenase (COX) targets and eicosanoid products in tumor cell growth. Sulindac sulfide, a COX inhibitor, inhibited the growth of non-small-cell lung cancers (NSCLC) both in soft agar and as xenografts in nude mice. Importantly, the concentration of sulindac sulfide required to inhibit NSCLC cell growth greatly exceeded the concentration required to inhibit prostaglandin (PG) E 2 synthesis in NSCLC cells, suggesting that NSAID inhibition of cell growth is mediated by additional targets distinct from COX. Both sulindac sulfide and ciglitazone, a defined peroxisome proliferator-activated receptor-␥ (PPAR␥) agonist, stimulated a promoter construct containing a PPAR response element linked to luciferase and potently inhibited NSCLC cell growth at similar concentrations, indicating a role for PPAR␥ as a target of NSAID action in these cells. Overexpression of PPAR␥ in NSCLC cells strongly inhibited the transformed growth properties of the cells, providing a molecular confirmation of the results obtained with the PPAR␥ agonists. Increased expression of PPAR␥, as well as ciglitazone and sulindac sulfide induced expression of E-cadherin, which has been linked to increased differentiation of NSCLC. Despite the fact that SCLC cell lines expressed little or no cytosolic phospholipase A 2 , COX-1, or COX-2, sulindac sulfide and PPAR␥ agonists also inhibited the transformed growth of these lung cancer cells. We propose that PPAR␥ serves as a target for NSAIDs that accounts for COX-independent inhibition of lung cancer cell growth.