Hematoporphyrin ethers--III. Cellular uptake and photosensitizing properties

1. The cellular uptake and the efficiency in sensitizing cells to photoinactivation were determined for hematoporphyrin (Hp) diphenyl ether, Hp dicyclohexyl ether and Hp dihexyl ether. 2. The phenyl diether was taken up by the cells to the same degree as was the clinically used porphyrin preparation photofrin II, while the dihexyl and notably the dicyclohexyl ether were taken up 3-4 times better. 3. Furthermore, the quantum yields for photoinactivation of cells were similar for the three diethers and twice as large as that for photofrin II. 4. Fluorescence- and absorption spectroscopy indicate that these findings are related to the fact that photofrin II is much more aggregated in the cells than are the three Hp diethers. 5. When cells loaded with the porphyrins are incubated with porphyrin-free medium containing serum a certain percentage of the cell-bound drug is removed: 14% for photofrin II, 28% for Hp diphenyl ether, 50% for Hp dicyclohexyl ether and 20% for Hp dihexyl ether. 6. With respect to cell uptake and retention of the dyes, the data did not show any uniform relationship to the polarity of the drugs, in contrast to what has been found earlier for Hp diethers of linear hydrocarbons.     

Inhibition of lecithin-cholesterol acyltransferase by diphytanoyl phosphatidylcholine

Model high density lipoproteins containing human apolipoprotein A-I, cholesterol, and a variety of phosphatidylcholines (PCs) have been prepared and tested. The PCs included 1-palmitoyl-2-oleoyl PC (POPC) and its diether analog 1-O-hexadecyl-2-oleyl PC (POPC ether), 1,2-diphytanoyl PC (DPhPC), 1-palmitoyl-2-phytanoyl PC, and 1-phytanoyl-2-palmitoyl PC. All ester PCs were good acyl donors for the transesterification of cholesterol catalyzed by human lecithin-cholesterol acyltransferase except DPhPC, which showed no reactivity. The PCs containing one phytanoyl chain donated an acyl chain to cholesterol as fast as non-branched fatty acyl chains. However, the competitive inhibition of lecithin-cholesterol acyltransferase by POPC ether and DPhPC was similar, and both lipids formed a macromolecular matrix that supported the reactivity of other ester PC substrates. The bulk of physicochemical properties of model high density lipoproteins composed of DPhPC were indistinguishable from those of POPC ether. These properties included 1) alpha-helical content of the apoprotein as assessed by circular dichroism, 2) microviscosity as determined from the fluorescence polarization and lifetime of the probe 1,6-diphenyl-1,3,5-hexatriene, 3) macromolecular weight based upon analytical gel filtration chromatography, and 4) surface polarity revealed by the fluorescence of 6-propionyl-2(dimethylamino)naphthalene. The only major difference in a physicochemical property was that the molecular surface area of DPhPC (area = 69 A2 at collapse pressure) determined by monolayer methods was 17 A2 greater than that of POPC (area = 53 A2 at collapse pressure) at all surface pressures measured. We suggest that the properties of DPhPC in being enzymatically nonreactive but a competitive inhibitor are due to its much larger size and that the active site of lecithin-cholesterol acyltransferase cannot bind phospholipid substrates in a catalytically productive way if they have surface areas of 70 A2 or more.     

Intramolecular exciplexes based on benzoxazole: photophysics and applications as fluorescent cation sensors.

Exciplex behaviour of three benzoxazole derivatives has been detected and intensively investigated by means of steady-state and time-resolved fluorescence techniques and transient absorption spectroscopy. The fluorescence of these compounds shows the properties which are typical for the excited state charge transfer complexes (exciplexes). Besides of the short wavelength fluorescence, which is similar in spectral distribution to the fluorescence of the electron acceptor (2-p-tolyl-benzoxazole), the red shifted, broad and structureless emission band is observed in solvents of low and medium polarity. The detailed analysis of the fluorescence data shows that the ratio of the CT and LE fluorescence initially increases with increasing solvent polarity, achieves a maximum, and drops for more polar solvents (epsilon(s) = 7). Similar behaviour is observed for the exciplex fluorescence lifetimes. The overall fluorescence and the relative intersystem crossing quantum yields show the decrease of these values with increasing solvent polarity. These observations have been explained on the basis of Marcus-type theory for nonradiative charge transfer rate constants. Increasing solvent polarity strongly accelerates the back electron transfer process which recovers the whole molecule in the ground state. The probability of the compact exciplex formation (i.e. sandwich-type structures) depends on solvent viscosity and degree of freedom of the bending of the saturated linker. The compound containing crown ether as a donor subunit may be used as a fluorescent indicator of inorganic cations (barium and lithium). We found an effective complexation of the compound in the ground state with barium and lithium cations. The complex is also stable in the excited state which manifests itself in strong increase of the fluorescence intensity.     

Spatial organization of axonal microtubules

Several workers have found that axonal microtubules have a uniform polarity orientation. It is the "+" end of the polymer that is distal to the cell body. The experiments reported here investigate whether this high degree of organization can be accounted for on the basis of structures or mechanisms within the axon. Substantial depolymerization of axonal microtubules was observed in isolated, postganglionic sympathetic nerve fibers of the cat subjected to cold treatment; generally less than 10% of the original number of microtubules/micron 2 remained in cross section. The number of cold stable MTs that remained was not correlated with axonal area and they were also found within Schwann cells. Microtubules were allowed to repolymerize and the polarity orientation of the reassembled microtubules was determined. In fibers from four cats, a majority of reassembled microtubules returned with the original polarity orientation. However, in no case was the polarity orientation as uniform as the original organization. The degree to which the original orientation returned in a fiber was correlated with the number of cold-stable microtubules in the fiber. We suggest that stable microtubule fragments serve as nucleating elements for microtubule assembly and play a role in the spatial organization of neuronal microtubules. The extremely rapid reassembly of microtubules that we observed, returning to near control levels within the first 5 min, supports microtubule elongation from a nucleus. However, in three of four fibers examined this initial assembly was followed by an equally rapid, but transient decline in microtubule number to a value that was significantly different than the initial peak. This observation is difficult to interpret; however, a similar transient peak has been reported upon repolymerization of spindle microtubules after pressure induced depolymerization.     

Insect epidermis: disturbance of supracellular tissue polarity does not prevent the expression of cell polarity

Summary The insect integument displays planar tissue polarity in the uniform orientation of polarized cuticular structures. In a body segment, for example, the denticles and bristles produced by the constituent epidermal cells point posteriorly. Colchicine can abolish this uniform orientation while still allowing individual cells to form orientated cuticular structures and thereby to express cell polarity. This suggests that an individual cell in a sheet can establish planar polarity without reference to some kind of covert supracellular cue (such as a morphogen gradient) in the epidermis as a whole. The results also indicate that colchicine interferes — directly or indirectly — with the mechanisms involved in aligning the polarity axes of individual cells into a common orientation, thereby generating supracellular or tissue polarity.     

Mutagenicity and chemical analysis of sequential organic extracts of airborne particulates

To obtain insight into the identity of chemicals associated with the mutagenicity of United States National Institute of Standards and Technology (NIST) Standard Reference Materials SRM 1649 (urban dust) and SRM 1650 (diesel particulate), parallel mutagenicity tests and chemical analyses were performed on dichloromethane and sequential organic extracts of these samples. SRM 1649 and 1650 were sequentially extracted with five organic solvents of increasing polarity, in order to partition mutagenic components into discrete fractions. The solvents (with associated polarity index) were as follows: (1) hexane (0.0); (2) hexane:diethyl ether 9:1 (0.29); (3) hexane:diethyl ether 1:1 (1.45); (4) diethyl ether (2.9); (5) methanol (6.6). 0.9270 g of SRM 1649, and 0.0510 g of SRM 1650 were each extracted three times with 8 ml of each of the solvents, the three aliquots were pooled, and analysed for target organics or solvent-exchanged into DMSO for mutagenicity testing in Salmonella typhimurium strains TA98 and TA100.The dichloromethane extracts of SRM 1649 and SRM 1650 contained direct-actin mutagens in Salmonella strains TA98 and TA100; SRM 1650 was significantly more potent than SRM 1649 in either strain. Addition of S9 caused a large decrease in mutagenicity of each extract, although SRM 1650 remained more potent. An interesting pattern of mutagenicity was observed for the sequential extracts of SRM 1649 and SRM 1650: the mutagenic potency of SRM 1649 extracts increased with increasing polarity of the extraction solvent while the response of the SRM 1650 extracts was the opposite. This suggests that the direct-acting mutagens in SRM 1650 are unlike those in SRM 1649. The response, though diminished, was largely unchanged when S9 was included in the test mixture.Chemical analyses on the various extracts were performed using a Hewlett-Packard model 5890 gas chromatograph equipped with a model 5970B mass selective detector (GC-MSD), and a 0.3 μm film thickness cross-linked methyl silicone capillary column (HP 1909A-101). Selected ion monitoring (SIM) methods were used to analyze for 105 target compounds including PAHs and nitro-PAHs. Chemical analysis of the dichloromethane extracts of SRM 1649 and SRM 1650 identified three main classes of compounds: polyaromatic hydrocarbons (PAH), vitro-polyaromatic hydrocarbons (NO₂-PAHs) and heterocyclics. The concentration of target compounds and the proportion of vitro-PAHs and heterocyclic compounds were considerably greater in SRM 1650 than in SRM 1649, consistent with the observed differences in their mutagenic potency. However, the different responses of the dichloromethane extracts in TA98 and TA100 suggest the presence of different (unidentified) compounds.Many of the target compounds were detected at least once in the sequential extracts from SRM 1649 and SRM 1650. There was no evident relationship between the occurrence of extracted organics, or classes of organics, and the polarity of solvents, except that, generally, the largest amount and variety of compounds were recovered in the first and second extracts (hexane; hexane:diethyl ether, 9:1). Preliminary examination of the chemical analysis results did not provide an explanation of the observed trends in mutagenic response. No single class of chemicals or individual compound was found to account for the observed pattern of mutagenicity. Compounds other than those identified must also contribute to the observed mutagenicity of any of the SRM 1649 and SRM 1650 extracts.     

Remnant lipoproteins induced proliferation of human prostate cancer cell,PC-3 but not LNCaP,via low density lipoprotein receptor

Background: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. Methods: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. Results: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. Conclusions: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.     

Thermotropic structural rearrangements in blood plasma lipoproteins in ischemic heart disease

Structural properties of blood plasma lipoproteins (LP) from coronary heart disease (CHD) patients were examined. Substantial differences were found in the pattern of the heat-induced structural restitution of LP of all classes in health and disease. At the same time no such differences were discovered in lipids isolated from LP. In high density lipoproteins from the plasma of CHD patients, a decrease in the microenvironmental polarity was shown to take place at a depth of 8 A from the surface of LP. The data obtained indicate substantial changes in the structural organization of LP of all classes in CHD. It is assumed that these changes are consequent on the modification of apoproteins and/or protein-lipid interactions in LP.     

Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.     

Wnt/PCP signaling: a veritable polar star in establishing patterns of polarity in embryonic tissues

Embryonic patterning has traditionally been viewed as the establishment of spatially significant gene expression in response to secreted signals. Recent work has highlighted the role of the Wnt/planar cell polarity (PCP) pathway in patterning tissues. Rather than establishing characteristic arrays of gene expression, however, this pathway functions to institute uniform polarity of cells within a tissue. Cells thus polarized can undergo directed migrations, cell divisions, etc., which are essential for normal morphogenesis. In this review, I will highlight the similarities between mechanisms that establish patterns of polarity between Drosophila and vertebrates. Further, I will discuss recent advances with regard to Wnt/PCP signaling in vertebrates.     

Lipoproteins, macrophage function, and atherosclerosis: beyond the foam cell?

Atherogenesis requires and is highly influenced by the interaction between lipoproteins and macrophages. Most of the focus to date has been on the ability of atherogenic lipoproteins (such as low-density lipoproteins, LDL) to promote and of anti-atherogenic lipoproteins (such as high-density lipoproteins, HDL) to prevent the development of the cholesteryl ester-enriched macrophage-derived foam cell. However, lipoprotein-macrophage interactions have the potential to modulate macrophage function in a variety of additional ways that may impact on atherosclerosis. These include modulating cellular cholesterol and oxysterol content, providing fatty acids as ligands for PPARs, and acting as ligands for macrophage scavenger and Toll-like receptors. We suggest that atherogenic lipoproteins promote and anti-atherogenic lipoproteins inhibit atherogenesis by modulating macrophage function in a variety of ways beyond cholesteryl ester accumulation and foam cell formation.     

Validation of a procedure for exogenous isotopic labeling of lipoprotein triglyceride with radioactive triolein.

A procedure has been developed for the exogenous isotopic labeling of triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) using high specific activity radioactive triglyceride in the presence of aqueous dimethyl sulfoxide. The labeled product lipoproteins showed unchanged chemical and physical properties. When the particles had also been labeled biologically by incorporation of unesterified fatty acids into the triglycerides of lipoproteins secreted by liver or intestine, both endogenous and exogenous labels were removed at the same rates in the isolated perfused heart and liver or in intact or functionally hepatectomized rats. These experiments additonally indicated that the triglyceride fatty acid composition of chylomicrons and very low density lipoproteins was unchanged during triglyceride depletion in the peripheral tissues. Using such labeled lipoproteins it has been shown that uptake of remnant lipoprotein cholesteryl ester and triglyceride by the liver is simultaneous. The labeling procedure described should prove suitable for kinetic studies of the disposition of the various lipoprotein non-polar ('core') lipids.     

Toll-like receptors: lessons from knockout mice

The Toll signalling pathway, which is required for establishment of dorsoventral polarity in Drosophila embryos, plays an important role in the response to microbial infections. Recently, Toll-like receptors (TLRs) have also been identified in mammals. TLR4 has been shown to function as the transmembrane component of the lipopolysaccharide receptor, while TLR2 recognizes peptidoglycans from Gram-positive bacteria, lipoproteins and yeast. Although various microbial cell-wall components are recognized by different receptors, all of these responses are abrogated in MyD88-deficient cells. These results show that different TLRs recognize different microbial cell-wall components, and that MyD88 is an essential signalling molecule shared among interleukin-1 receptor/Toll family members.     

Interaction between haptoglobin subtypes and AB0 blood groups in a Bengalee population

Blood samples from 621 individuals of a Caste Hindu Population from West Bengal (India) were investigated in an attempt to find out an association between the AB0 blood groups and Haptoglobin (HP) subtypes. AB0 blood grouping was done on the basis of the agglutination test with standard anti-sera. Haptoglobin subtyping only for the HP*1 allele was done by Polyacrylamide Gel Electrophoresis (PAGE). A significant association was found with a significantly lower HP*1S allele frequency in blood group 0 versus other AB0 blood groups. A comparatively higher allele frequency of HP*1S was found in this population sample. An inverse relationship between HP*1S and HP*2 has been revealed in each blood group. It appears that the major portion of HP*1 alleles in the A, B, and AB blood groups belongs to the HP*1S allele compared to that of the 0 blood group.     

Ear oximetry during combined hypoxia and exercise

Ear oximetry is widely used to detect arterial O2 desaturation during exercise in patients with cardiopulmonary disease. Although oximeters have been evaluated for accuracy, response time, and the influence of skin pigmentation, tests of their reliability have not been reported during strenuous exercise. Accordingly, we compared arterial O2 saturation (Sao2) measurements obtained by Hewlett-Packard (HP, model 47201A) and Biox II oximeters with those determined directly from arterial blood in six healthy volunteers during progressive exercise while rebreathing hypoxic gas mixtures. The relationship between the HP oximeter value and blood Sao2 was described by the equation: HP = 0.93 (Sao2) + 5.37 and for the Biox II: Biox = 0.55 (Sao2) + 38.97. With these equations, at a blood Sao2 value of 90%, the underestimation by both oximeters was less than 2%. At a blood value of 70%, the HP oximeter overestimated blood Sao2 by 0.7%, whereas the Biox II showed an overestimation of 10.7%. Below blood Sao2 of 83%, the Biox II tended to overestimate blood Sao2 by an amount greater than the error of the instrument, whereas the HP estimations were within the error of the instrument over all levels of blood Sao2 studied. We conclude that the HP oximeter provides valid estimates of Sao2 during exercise but that the Biox II oximeter, although reflecting qualitative changes in oxygenation that occur during exercise, does not provide accurate records of the degree of desaturation.     

Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity

VapD-like virulence-associated proteins have been found in many organisms, but little is known about this protein family including the 3D structure of these proteins. Recently, a relationship between the Cas2 family of ribonucleases associated with the CRISPR system of microbial immunity and VapD was suggested. Here, we show for the first time the structure of a member of the VapD family and present a relationship of VapD with Cas2 family and toxin–antitoxin (TA) systems. The crystal structure of HP0315 from Helicobacter pylori was solved at a resolution of 2.8 Å. The structure of HP0315, which has a modified ferredoxin-like fold, is very similar to that of the Cas2 family. Like Cas2 proteins, HP0315 shows endoribonuclease activity. HP0315-cleaved mRNA, mainly before A and G nucleotides preferentially, which means that HP0315 has purine-specific endoribonuclease activity. Mutagenesis studies of HP0315 revealed that D7, L13, S43 and D76 residues are important for RNase activity, in contrast, to the Cas2 family. HP0315 is arranged as an operon with HP0316, which was found to be an antitoxin-related protein. However, HP0315 is not a component of the TA system. Thus, HP0315 may be an evolutionary intermediate which does not belong to either the Cas2 family or TA system.     

Mutagenicity and chemical analysis of sequential organic extracts of airborne particulates.

To obtain insight into the identity of chemicals associated with the mutagenicity of United States National Institute of Standards and Technology (NIST) Standard Reference Materials SRM 1649 (urban dust) and SRM 1650 (diesel particulate), parallel mutagenicity tests and chemical analyses were performed on dichloromethane and sequential organic extracts of these samples. SRM 1649 and 1650 were sequentially extracted with five organic solvents of increasing polarity, in order to partition mutagenic components into discrete fractions. The solvents (with associated polarity index) were as follows: (1) hexane (0.0); (2) hexane:diethyl ether 9:1 (0.29); (3) hexane:diethyl ether 1:1 (1.45); (4) diethyl ether (2.9); (5) methanol (6.6). 0.9270 g of SRM 1649, and 0.0510 g of SRM 1650 were each extracted three times with 8 ml of each of the solvents, the three aliquots were pooled, and analysed for target organics or solvent-exchanged into DMSO for mutagenicity testing in Salmonella typhimurium strains TA98 and TA100. The dichloromethane extracts of SRM 1649 and SRM 1650 contained direct-acting mutagens in Salmonella strains TA98 and TA100; SRM 1650 was significantly more potent than SRM 1649 in either strain. Addition of S9 caused a large decrease in mutagenicity of each extract, although SRM 1650 remained more potent. An interesting pattern of mutagenicity was observed for the sequential extracts of SRM 1649 and SRM 1650: the mutagenic potency of SRM 1649 extracts increased with increasing polarity of the extraction solvent while the response of the SRM 1650 extracts was the opposite. This suggests that the direct-acting mutagens in SRM 1650 are unlike those in SRM 1649. The response, though diminished, was largely unchanged when S9 was included in the test mixture. Chemical analyses on the various extracts were performed using a Hewlett-Packard model 5890 gas chromatograph equipped with a model 5970B mass selective detector (GC-MSD), and a 0.3 microns film thickness cross-linked methyl silicone capillary column (HP 1909A-101). Selected ion monitoring (SIM) methods were used to analyze for 105 target compounds including PAHs and nitro-PAHs. Chemical analysis of the dichloromethane extracts of SRM 1649 and SRM 1650 identified three main classes of compounds: polyaromatic hydrocarbons (PAH), nitro-polyaromatic hydrocarbons (NO2-PAHs) and heterocyclics. The concentration of target compounds and the proportion of nitro-PAHs and heterocyclic compounds were considerably greater in SRM 1650 than in SRM 1649, consistent with the observed differences in their mutagenic potency. However, the different responses of the dichloromethane extracts in TA98 and TA100 suggest the presence of different (unidentified) compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transition state stabilization of subtilisins in organic media

Electrostatic forces are among the stabilizing interactions that contribute to the high degree of enzyme-transition state complementarity. The active-site polarity, which can differ substaintially from that of water, is thus an important determinant of transition state stabilization. Here we pose the question of whether the rate of an enzymatic reaction proceeding through a charged transition state can be increased by increasing the active-site polarity in an organic solvent. The active-site polarity of subtilisin has been reduced by dehydration and suspension in a nonpolar solvent (tetrahydrofuran), and then increased by adding water to the solvent. Enhancing the local polarity substantially increasing the rate of catalysis, implicating polarity as an important factor in stabilizing the charged tetrahedral transition state. Studies with subtilisins whose active sites have been modified by site-directed mutagenesis support the role of polarity in transition state stabilization. (c) 1994 John Wiley & Sons, Inc.     

The class B, type I scavenger receptor promotes the selective uptake of high density lipoprotein cholesterol ethers into caveolae

The uptake of cholesterol esters from high density lipoproteins (HDLs) is characterized by the initial movement of cholesterol esters into a reversible plasma membrane pool. Cholesterol esters are subsequently internalized to a nonreversible pool. Unlike the uptake of cholesterol from low density lipoproteins, cholesterol ester uptake from HDL does not involve the internalization and degradation of the particle and is therefore termed selective. The class B, type I scavenger receptor (SR-BI) has been identified as an HDL receptor and shown to mediate selective cholesterol ester uptake. SR-BI is localized to cholesterol- and sphingomyelin-rich microdomains called caveolae. Caveolae are directly involved in cholesterol trafficking. Therefore, we tested the hypothesis that caveolae are acceptors for HDL-derived cholesterol ether (CE). Our studies demonstrate that in Chinese hamster ovary cells expressing SR-BI, >80% of the plasma membrane associated CE is present in caveolae after 7.5 min of selective cholesterol ether uptake. We also show that excess, unlabeled HDL can extract the radiolabeled CE from caveolae, demonstrating that caveolae constitute a reversible plasma membrane pool of CE. Furthermore, 50% of the caveolae-associated CE can be chased into a nonreversible pool. We conclude that caveolae are acceptors for HDL-derived cholesterol ethers, and that caveolae constitute a reversible, plasma membrane pool of cholesterol ethers.     

Assessment of heat production, heat loss, and core temperature during nitrous oxide exposure: a new paradigm for studying drug effects and opponent responses

Studies using core temperature (T(c)) have contributed greatly to theoretical explanations of drug tolerance and its relationship to key features of addiction, including dependence, withdrawal, and relapse. Many theoretical accounts of tolerance propose that a given drug-induced psychobiological disturbance elicits opponent responses that contribute to tolerance development. This proposal and its theoretical extensions (e.g., conditioning as a mechanism of chronic tolerance) have been inferred from dependent variables, such as T(c), which represent the summation of multiple underlying determinants. Direct measurements of determinants could increase the understanding of opponent processes in tolerance, dependence, and withdrawal. The proximal determinants of T(c) are metabolic heat production (HP) and heat loss (HL). We developed a novel system for simultaneously quantifying HP (indirect calorimetry), HL (direct gradient layer calorimetry), and T(c) (telemetry) during steady-state administrations of nitrous oxide (N(2)O), an inhalant with abuse potential that has been previously used to study acute and chronic tolerance development to its hypothermia-inducing property. Rats were administered 60% N(2)O (n = 18) or placebo gas (n = 16) for 5 h after a 2-h placebo baseline exposure. On average, N(2)O rapidly but transiently lowered HP and increased HL, each by approximately 16% (P < 0.001). On average, rats reestablished and maintained thermal equilibrium (HP = HL) at a hypothermic T(c) (-1.6 degrees C). However, some rats entered positive heat balance (HP > HL) after becoming hypothermic such that acute tolerance developed, i.e., T(c) rose despite continued drug administration. This work is the first to directly quantify the thermal determinants of T(c) during administration of a drug of abuse and establishes a new paradigm for studying opponent processes involved in acute and chronic hypothermic tolerance development.