25-hydroxycholesterol increases eicosanoids and alters morphology in cultured pulmonary artery smooth muscle and endothelial cells.

25-hydroxycholesterol (25-OHC) is an oxidized derivative of cholesterol that has been implicated in the early development of arteriosclerosis. Changes in arterial smooth muscle cell (SMC) migration and proliferation have also been linked to the pathophysiology of arteriosclerosis. SMCs undergo "activation" in response to vascular injury by changing phenotypically and by increasing prostaglandin G/H synthase-2 (PGHS-2) protein levels and eicosanoid release. Activation is thought to be important in atheroma formation and arteriosclerosis progression. 25-OHC induces SMCs to change morphologically, increase PGHS-2, and increase eicosanoid release. Confluent monolayers were treated with 25-OHC (10 microg/mL) or the PGHS-2 inducer interleukin-1beta (1 ng/mL) for 18 hours at 37 degrees C. The 18-hour treatment resulted in morphological changes. After uptake of [(14)C]arachidonic acid, released radiolabeled arachidonic acid products were extracted and chromatographed by both normal and reverse-phase high-performance liquid chromatography systems. 25-OHC-treated cells increased their prostaglandin production, with the major component comigrating with a prostaglandin-E(2) standard. HETEs and epoxyeicosatrienoic acids were not affected. Immunoprecipitation analysis of treated and control cell lysates using anti-PGHS-1 and -2 and anti-alpha-actin primary antibodies indicated PGHS-2 induction over control and no change in contractile proteins. These changes are consistent with SMC activation, which occurs in vascular injury models. The notion that oxysterols can activate vascular SMCs may be important in ultimately understanding the pathophysiology of atheroma formation.

Raman microspectroscopy of intracellular cholesterol crystals in cultured bovine coronary artery endothelial cells.

Raman microspectroscopy is presented as a promising technique for the in situ characterization of intracellular cholesterol crystals. Crystal characterization is the first step in investigating the effects of various stimuli on their formation and in determining their role in the development of atherosclerosis. Treatment of cultured bovine coronary artery endothelial cells with 22-hydroxycholesterol (220HC) stimulated the production of intracellular crystals, a phenomenon that did not occur in the absence of viable cells. These crystals were identified as a combination of the 220HC starting material and cholesterol. The best fit to the average Raman spectrum of the microscopic crystals was achieved with a combination of 70% Raman contribution from 220HC and 30% from cholesterol. GC/MS analysis of the crystals confirmed the presence of both compounds. These results demonstrate the potential of Raman microspectroscopy as a powerful tool in lipid research, particularly for the in situ characterization of intracellular crystals.

25-Hydroxycholesterol enhances eicosanoid production in cultured bovine coronary artery endothelial cells by increasing prostaglandin G/H synthase-2.

Oxidized derivatives of cholesterol, the oxysterols, have been implicated in the development of atherosclerosis. We have found that the oxysterol, 25-hydroxycholesterol (25OHC), is a potent stimulator of eicosanoid production in endothelial cells. Confluent monolayers of bovine coronary artery endothelial cells (BCAECs) were treated with 25OHC (10 micrograms/ml) or interleukin-1 beta (IL-1 beta, 1 ng/ml), a known prostaglandin G/H synthase-2 (PGHS-2) inducer, for 48 h at 37 degrees C. Following incubation with [14C]arachidonic acid, the 14C-labeled metabolites of arachidonic acid were extracted and resolved using both normal and reverse phase high-performance liquid chromatography (HPLC) systems. 25OHC-treated cells had a five-fold increase in their prostaglandin production when compared to untreated cells. Eicosanoid production in IL-1 beta treated cells was not as pronounced. The major component in both sets of cells comigrated with 6-ketoPGF1 alpha. Other PGHS metabolites, 15- and 11-hydroxyeicosatetraenoic acids (HETEs) and 12-hydroxyheptadecatrienoic acid (HHT) were also identified and increased following 25OHC treatment. Epoxyeicosatrienoic acids, metabolites of cytochrome P450, were not effected. Enhanced production of 6-ketoPGF1 alpha, 15-HETE, 11-HETE and HHT were inhibited by indomethacin. Thus, all eicosanoids induced by 25OHC treatment were PGHS products. Western immunoblot analysis of lysates from 25OHC, IL-1 beta, or vehicle treated cells using anti-PGHS-1 and -2 antibodies revealed a significant increase in PGHS-2, but not PGHS-1, in both 25OHC and IL-1 beta treated cells. The notion that oxysterols can modulate vascular eicosanoid production through enzyme induction may be important in ultimately understanding their role in the pathogenesis of atherosclerosis.

Synthesis and characterization of a heterobifunctional mercurial cross-linking agent: incorporation into cobratoxin and interaction with the nicotinic acetylcholine receptor.

The heterobifunctional organomercurial reagents 3-(acetoxymercurio)- and 3-(chloromercurio)-5-nitrosalicylaldehyde were prepared, characterized in model studies, and used to probe the interaction between cobratoxin, purified from the venom of the Thailand cobra (Naja naja siamensis), and the affinity-purified nicotinic acetylcholine receptor (AcChR) from Torpedo california electroplax. These reagents may also be useful in introducing chemically well-defined heavy metal atoms into proteins containing no reactive thiols. Model reagent adducts were prepared in situ by reductive amination with N-butylamine and N alpha-acetyllysine-N-methylamide. The nitrophenolic pKaS of the amine adducts were similar to those of the aldehyde reagents through reduced by 1.3-1.5 units when compared with the hydroxylmethyl reduction product. Reaction of either mercuriosalicylaldehyde with cobratoxin led to a single major modification product incorporating 1 mol of the reagent into cobratoxin at Lys 23. The Lys 23 modified toxin had a reduced binding affinity for the AcChR over that of the native toxin (Kd 2.75 nM cf. 0.3 nM). Reduction of the purified AcChR with 1 mM dithiothreitol (DTT) followed by removal of excess thiol led to cross-linking reactions with the Lys 23 modified cobratoxin to both the alpha and beta subunits of the AcChR complex. Reaction of DTT-treated AcChR with N-ethylmaleimide (NEM) blocked cross-linking, while treatment of the initially cross-linked toxin-AcChR complex with mercaptoethanol leads to reversal of cross-linking. These observations strongly support cross-linking mediated by the formation of a mercury-sulfur bond and further lend support the identity of the respective interacting sites in AcChR and toxin.

Redox reactive reagents inhibiting and inactivating choline acetyltransferase.

3-Trimethylammoniomethyl catechol and N,N-dimethylepinephrine (catecholine) are redox reactive reagents which possess quaternary ammonium functional groups and the capacity to inhibit or inactivate choline binding macromolecules which mediate cholinergic neuronal function. Earlier studies reported the synthesis of 3-trimethylammoniomethyl catechol and demonstrated its redox-dependent covalent inactivation of the nicotinic acetylcholine receptor (Nickoloff et al., Biochemistry 24, 999-1007 (1985)]. Here we present the synthesis of catecholine and show that both 3-trimethylammoniomethyl catechol and catecholine are weak noncompetitive inhibitors (Ki = 15 +/- 6 and 25 +/- 4 mM, respectively) of choline acetyltransferase (EC 2.3.1.6). Both agents irreversibly inactivate the enzyme.