Leukotriene B4 (LTB4), a potent leukocyte chemoattractant derived from the 5-lipoxygenase metabolism of arachidonic acid, exerts its action by means of specific cell surface receptors, denoted BLT1 and BLT2. In this study, BLT1 receptor proteins were detected in human carotid artery atherosclerotic plaques, colocalizing with markers for macrophages, endothelial cells, and vascular smooth muscle cells (SMC). Challenge of human coronary artery SMC with either LTB 4 or U75302, a partial agonist that is selective for the BLT1 receptor, induced an Ϸ4-fold increase of whole-cell currents by using the patch-clamp technique, indicating that these cells express functional BLT1 receptors. LTB4 induced migration and proliferation of SMC in vitro, and treatment with the BLT receptor antagonist BIIL 284 (10 mg͞kg, once daily) for 14 days after carotid artery balloon injury in vivo inhibited intimal hyperplasia in rats. In the latter model, SMC derived from the intima exhibited increased levels of BLT 1 receptor mRNA compared with medial SMC. BLT receptor up-regulation in the intima in vivo, as well as that induced by IL-1␤ in vitro, were prevented by transfection with a dominantnegative form of I kinase ␤ carried by adenovirus, indicating that BLT1 receptor expression depends on NF-〉. These results show that LTB4 activates functional BLT1 receptors on vascular SMC, inducing chemotaxis and proliferation, and that BLT1 receptors were up-regulated through an I kinase ␤͞NF-B-dependent pathway. Inhibition of LTB4͞BLT1 signaling during the response to vascular injury reduced intimal hyperplasia, suggesting this pathway as a possible target for therapy.restenosis ͉ cell proliferation ͉ chemotaxis ͉ lipoxygenase ͉ eicosanoids L eukotrienes (LTs) are inflammatory mediators that are derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism (1). Leukotrienes exert their actions by means of membrane-bound G protein-coupled receptors; CysLT receptors, which are activated by leukotrienes C 4 , D 4 , and E 4 ; and BLT receptors, which are activated by leukotriene B 4 (LTB 4 ) (2, 3). The last class consists of two receptor subtypes, BLT 1 and BLT 2 , representing the high-and low-affinity receptors for LTB 4 , respectively (4, 5). BLT receptors are expressed on leukocytes; neutrophils, eosinophils, and T lymphocytes all migrate in response to LTB 4 (6, 8-11).A major role for the leukotriene pathway in vascular disease was suggested by studies of a congenic mouse strain demonstrating resistance to atherosclerosis linked to a locus on chromosome 6, within which the gene for 5-LO was mapped subsequently (12). Also, expression of the enzymes and receptors of the 5-LO pathway has been demonstrated in human atherosclerotic plaques (13), and genetic studies have revealed that polymorphism in the 5-LO promoter is associated with an increased carotid artery intima thickness (14) and that the gene encoding the 5-LO-activating protein (FLAP) is associated with an increased risk of stroke and myocardial infarction (15).Although the...

Interaction of syntaxin 1 with the ␣ 1D subunit of the voltage-gated L type Ca 2؉ channel was investigated in the pancreatic ␤ cell. Coexpression of the enhanced green f luorescent protein-linked ␣ 1D subunit with the enhanced blue f luorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the ␣ 1D subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the ␣ 1D subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin We now show that syntaxin 1 colocalizes and associates with the ␣ 1D subunit of the voltage-gated L type Ca 2ϩ channel and thereby modulates not only Ca 2ϩ channel activity but also insulin release in a Ca 2ϩ -dependent manner. MATERIALS AND METHODSPreparation of Islets and Single ␤ cells. Islets of Langerhans and single pancreatic ␤ cells were isolated from adult obese mice (gene symbol ob͞ob) as described previously (11).Coexpression of ␣ 1D Subunit-Enhanced Green Fluorescent Protein (EGFP) and Enhanced Blue Fluorescent Protein (EBFP)-Syntaxin 1 and Fluorescence Microscopy. The cDNA for hamster ␣ 1D3a (provided by J. Dillon, New England Medical Center, Boston) was flanked with the human cytomegalovirus (CMV) promoter and the bovine growth hormone poly(A) site to enable expression in mammalian cells. A HindIII site was introduced by site-directed mutagenesis at the nucleotides coding for the last amino acid and the stop codon, which allowed in-frame fusion with the EGFP cDNA and generation of pCMV ␣ 1D3a EGFP. To create pCMV EBFPsyntaxin 1A, a SmaI site was introduced into the EBFP cDNA at the nucleotides coding for Asp-235 and Glu-237, generating pCMV EBFP0. The cDNA for rat syntaxin 1A (gift from R. H. Scheller, Stanford University, Stanford, CA) was removed from pRcCMV syntaxin 1A by digestion with EcoRV and XbaI and fused in-frame with the EBFP cDNA of the SmaI͞XbaI-opened pCMV EBFP0. Cultured single pancreatic ␤ cells were cotransfected with pCMV ␣ 1D3a EGFP and pCMV EBFPsyntaxin 1A overnight by the lipofectamine technique. The cotransfected cells were monitored for EGFP͞EBFP fluorescence 48 h after the start of transfection by using a Leica Fluovert FU microscope (Leica, Deerfield, IL) and PL Fluotar 100͞1.32 oil lens (Leitz) equipped with a LSR AstroCam TE3͞A͞S charge-coupled device camera (LSR, Cambridge, U.K.). Filter settings for EGFP fluorescence measurements were: excitation, HQ470͞40; dichroic mirror, Q495LP; and emission, HQ525͞50. Filter settings for EBFP fluorescence measurements were: excitation, D399͞22; dichroic mirror, 420DLCP; and emission, E430LP. Colocalization of ␣ 1D3a EGFP and EBFP-syntaxin 1A was studied by applying the deconvolution method on a stack of 15 images (200-nm vertical distance) using the iterative Tikhonov-Miller restoration procedure (Huygens System 2; Scientific Volume Imaging, Hilversum, The Netherlands).Density Gradient Subcellular Fractionation. Subcellular fractionation, using a linear sucrose gradient, was performed as described ...

Parafibromin is a protein product derived from the hyperparathyroidism 2(HRPT2) tumor suppressor gene and its inactivation has been coupled to familial and sporadic forms of parathyroid malignancy. In this study, we have conducted immunohistochemistry on 33 parathyroid carcinomas (22 unequivocal and 11 equivocal) using four parafibromin antibodies directed to different parts of the protein.Furthermore, for a fraction of cases, the immunohistochemical results were compared with known HRPT2 mutational status. Our findings show that 68% (15 out of 22) of the unequivocal carcinomas exhibited reduced expression of parafibromin while the 25 sporadic adenomas used as controls were entirely positive for parafibromin expression. Additionally, three out of the six carcinomas with known HRPT2 mutations showed reduced expression of parafibromin. Using all four antibodies, comparable results were obtained on the cellular level in individual tumors suggesting that there exists no epitope of choice in parafibromin immunohistochemistry. The results agree with the demonstration of a w60 kDa product preferentially in the nuclear fraction by western blot analysis. We conclude that parafibromin immunohistochemistry could be used as an additional marker for parathyroid tumor classification, where positive samples have low risk of malignancy, whereas samples with reduced expression could be either carcinomas or rare cases of adenomas likely carrying an HRPT2 mutation.