G-protein-coupled receptor dimerization: modulation of receptor function.

G-protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in the human genome that respond to a plethora of signals, including neurotransmitters, peptide hormones, and odorants, to name a few. They couple to second messenger signaling cascade mechanisms via heterotrimeric G-proteins. Recently, many studies have revealed that GPCRs exist as dimers, which may be present as homo- or heterodimers/oligomers. These recent findings have been met with skepticism, since they are contradictory to the dogma that GPCRs function as monomers. Although the existence of GPCR dimers/oligomers was predicted from early pharmacological and biochemical studies, further studies to critically evaluate this phenomenon were impeded by the lack of appropriate reagents. The availability of cDNAs for GPCRs, of highly selective ligands and of antibodies for these receptors has made it possible to visualize and investigate the functional effects of GPCR oligomers. Pharmacological studies, along with biochemical techniques, such as cross-linking and immunoprecipitation with differentially epitope-tagged receptors, have been employed to demonstrate the oligomerization of a number of GPCRs. Moreover, recent biophysical techniques, such as bioluminescence and fluorescence resonance energy transfer, now make it possible to examine GPCR dimerization/oligomerization in living cells. In this review, we provide a brief overview of some of the techniques employed to describe GPCR dimers, and we discuss their respective limitations. We also examine the implications of dimerization/oligomerization on GPCR function. In addition, we discuss domains of the receptors that are thought to facilitate dimerization/oligomerization. Finally, we consider recent evidence for the subcellular localization of the dimer/oligomer assembly.

Overexpression of human superoxide dismutase inhibits oxidation of low-density lipoprotein by endothelial cells.

Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O2.-) and oxidize LDL in vitro; however, the role of O2.- in endothelial cell-induced LDL oxidation is unclear. Incubation of human LDL (200 microg/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5+/-1.1 versus 6.3+/-0.2 [mean+/-SEM] nmol malondialdehyde/mg LDL protein, respectively; P<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 microg/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O2.- release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O2.- contributes to endothelial cell-induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells.

Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis.

The endothelium is a source of reactive oxygen species in short-term models of hypercholesterolemia and atherosclerosis. We examined a chronic model of atherosclerosis for increased vascular production of superoxide (O2-.) and determined whether endothelial overexpression of superoxide dismutase (SOD) would improve endothelium-dependent relaxation. Superoxide generation was 3 times higher in isolated aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits (2 to 4 years old) compared with aortas from New Zealand White (NZ) rabbits (43+/-10 versus 14+/-2 relative light units x min(-1) x mm(-2), n=9, P<0.05). After in vitro transduction with adenovirus containing the gene for CuZn-SOD (AdCMVCuZn-SOD) or extracellular SOD (AdCMVEC-SOD), endothelial O2-. levels in WHHL aortas were significantly reduced. Gene transfer of SOD to WHHL aortas, however, failed to improve the impaired relaxation to acetylcholine or calcium ionophore. By use of the oxidative fluorescent dye hydroethidine, an in situ assay indicated markedly increased generation of O2-. throughout the wall of WHHL aorta, especially within layers of smooth muscle. This finding was confirmed by demonstrating increased O2-. levels in smooth muscle cells cultured from WHHL aorta. We conclude that elevated O2-. levels in atherosclerotic vessels are not confined to the endothelium but occur throughout the vascular wall, including smooth muscle cells. Reduction in endothelial O2-. levels is not sufficient to improve endothelium-dependent relaxation. Generation of reactive oxygen species within the media may contribute to vasomotor dysfunction in atherosclerosis.

Augmented adenovirus-mediated gene transfer to atherosclerotic vessels.

Vascular endothelium is an important target for gene transfer in atherosclerosis. In this study, we examined gene transfer to normal and atherosclerotic blood vessels from two species, using an organ culture method. Using normal aorta, we determined optimal dose, duration of exposure to adenovirus, and duration of incubation of vessels in tissue culture. Aortas from normal and atherosclerotic monkeys were cut into rings and incubated for 2 hours with a recombinant adenovirus, carrying the reporter gene for beta-galactosidase driven by a cytomegalovirus (CMV) promoter. After 20 hours of incubation, transgene expression was assessed with a morphometric method after histochemical staining and a chemiluminescent assay of enzyme activity. Expression of beta-galactosidase after histochemical staining, expressed as percentage of total cells, was similar in adventitial cells of normal monkeys (21 +/- 4%, mean +/- SE%) and atherosclerotic monkeys (25 +/- 12%). Transgene expression in endothelium was higher in atherosclerotic than in normal vessel (53 +/- 3% versus 27 +/- 7%, P < .05). Chemiluminescent assay indicated greater beta-galactosidase activity (2.5 +/- 0.6 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.6 +/- 0.2 mU/mg of protein, P < .05). Aortas from normal (n = 6) and atherosclerotic (n = 5) rabbits also were examined. Transgene expression (after histochemical staining) in endothelium was much greater in atherosclerotic than normal rabbits (39 +/- 3% versus 9 +/- 2%, P < .05) and expression in adventitial cells was similar (normal 23 +/- 2%, atherosclerotic 24 +/- 4%). Chemiluminescent assay indicated greater beta-galactosidase activity (1.2 +/- 0.4 mU/mg of protein) in the intima and media of atherosclerotic than normal vessels (0.2 +/- 0.1 mU/mg protein, P < .05). These findings suggest that an adenoviral vector with a CMV promoter provides similar transgene expression in adventitia of both normal and atherosclerotic vessels. Gene transfer to the endothelium was much more effective in atherosclerotic than in normal vessels. Thus it may be possible to achieve greater transgene expression in atherosclerotic than in normal arteries.

Altered vascular function after adenovirus-mediated overexpression of endothelial nitric oxide synthase.

Gene transfer with replication-deficient adenovirus is a potentially useful tool to study vascular biology. We have constructed a replication-deficient adenovirus (AdRSVeNOS) that carries cDNA for endothelial nitric oxide synthase (eNOS). Transfection of COS-1 cells with AdRSVeNOS increased nitric oxide synthase activity (measured as production of L-citrulline from L-arginine) that was calcium dependent and inhibited by N omega-nitro-L-arginine methyl ester. To investigate effects of overexpression of eNOS on vascular function, we incubated common carotid arteries from rabbits in organ culture with AdRSVeNOS or AdRSV beta gal encoding beta-galactosidase. Transgene expression and responses to vasoactive agents were examined 1 day after transduction. Histochemical staining of beta-galactosidase and immunohistochemistry for eNOS indicated transgene expression in endothelium and adventitial cells. After precontraction with phenylephrine, vessels treated with AdRSVeNOS demonstrated greater relaxation to acetylcholine than vessels treated with vehicle or AdRSV beta gal. Relaxation to calcium ionophore A-23187 was much greater in vessels treated with AdRSVeNOS than in vessels treated with vehicle or AdRSV beta gal. Augmented relaxation in response to A-23187 was also observed after denudation of endothelium in vessels treated with AdRSVeNOS and was inhibited by N omega-nitro-L-arginine. Thus vasorelaxation in response to stimuli that release nitric oxide is augmented after adenovirus-mediated overexpression of eNOS. Transgene expression in adventitial cells appears to be sufficient to alter vasomotor function.

Novel methods for adenovirus-mediated gene transfer to blood vessels in vivo.

Adenovirus-mediated gene transfer is a promising method for studies of vascular biology and potentially for gene therapy. Intravascular approaches for gene transfer to blood vessels in vivo generally require interruption of blood flow and have several limitations. We have used two alternative approaches for gene transfer to blood vessels in vivo using perivascular application of vectors. First, replication-deficient adenovirus expressing nuclear-targeted bacterial beta-galactosidase was injected into cerebrospinal fluid via the cisterna magna of rats. Leptomeningeal cells over the major arteries were efficiently transfected, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. When viral suspension was injected with the rat in a lateral position, the reporter gene was expressed extensively on the ipsilateral surface of the brain. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, positioning of the head may 'target' specific regions of the brain. Second, vascular gene delivery was accomplished by perivascular injection of virus in peripheral vessels. Injection of the adenoviral vector within the periarterial sheath of monkeys resulted in gene transfer to the vessel wall that was substantial in magnitude although limited to cells in the adventitia. Approximately 20% of adventitial cells expressed the transgene, with no gene transfer to cells in the intima or media. These approaches may provide alternative approaches for gene transfer to blood vessels, and may be useful for studies of vascular biology and perhaps vascular gene therapy.

Intrapericardial administration of adenovirus for gene transfer.

Gene transfer to the heart has been accomplished with intravascular administration of adenoviral vectors into the pericardial sac, by increasing the duration of exposure to the adenovirus, would result in gene expression in the pericardium and perhaps myocardium and therefore might provide an alternative method to intravascular administration for gene transfer. We injected a replication-deficient adenovirus (average 1 x 10(12) particles/ml in 3% sucrose; 1 x 10(10) plaque forming units/ml containing cDNA encoding a nuclear-targeted bacterial beta-galactosidase into the pericardial sac of dogs. Samples of the pericardium and heart were examined for enzymatic activity of beta-galactosidase and after histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. One day after injection of the adenovirus (1-3 ml), beta-galactosidase activity was highest in the parietal pericardium and left atrial tissue and lower in the right and left ventricles. Histochemical expression of the transgene was predominantly in the visceral pericardium of atria and ventricles and occasionally in the epicardial myocytes, arterioles, and venules. Pretreatment with doxycycline (5 mg) before adenovirus administration increased transgene activity in left ventricles. Thus adenovirus injected into the pericardial sac provides an effective method for gene transfer to the visceral and parietal pericardium over atria and ventricles.

Vascular expression of inducible nitric oxide synthase is associated with activation of Ca(++)-dependent K+ channels.

We tested the hypothesis that expression of inducible nitric oxide synthase (NO-synthase) in response to endotoxin (lipopolysaccharide) produces activation of potassium channels. Contraction of the rat thoracic aorta in response to phenylephrine was measured in vitro after treatment in vivo for 15 hr with vehicle (control) or lipopolysaccharide (10 mg/kg i.p.). Impaired contraction in response to phenylephrine was used as an index of inducible NO-synthase expression, and activation of potassium channels was examined with specific inhibitors. Contraction in response to 10(-5) M phenylephrine (expressed as a percentage of contraction in response to 85 mM KCI) was markedly impaired in lipopolysaccharide-treated rats, compared with control (15 +/- 5% vs. 131 +/- 10%, P < .05, mean +/- S.E.). Expression of inducible NO-synthase mRNA in the vessel wall in lipopolysaccharide-treated rats was confirmed using reverse transcription-polymerase chain reaction. Contraction of the aorta in lipopolysaccharide-treated rats was restored to normal by 0.3 mM aminoguanidine (an inhibitor of inducible NO-synthase). Contraction of the aorta in response to phenylephrine, which was inhibited by lipopolysaccharide, was not affected by glibenclamide (an inhibitor of ATP-sensitive potassium channels) but was increased 2-fold (P < .05) by iberiotoxin (50 nM), an inhibitor of Ca(+2)-dependent potassium channels. Relaxation of the aorta in response to sodium nitroprusside, an exogenous donor of nitric oxide, and 8-bromo-cyclic GMP was also inhibited by iberiotoxin. These findings suggest that nitric oxide produced by vascular expression of inducible NO-synthase activates calcium-dependent potassium channels and that this mechanism may contribute to impaired vasoconstrictor responses during sepsis.

Adenovirus-mediated gene transfer to normal and atherosclerotic arteries. A novel approach.

Previous studies of gene transfer to blood vessels in vivo have relied on intraluminal, catheter-based methods for delivery of adenoviral and other vectors. In this study, topical application of a replication-deficient adenoviral vector was used as an alternative method of gene transfer to the vessel wall. We administered recombinant adenovirus (1.0 x 1.5 x 10(10) pfu/mL) containing the nuclear targeted bacterial beta-galactosidase gene topically to arteries in normal and atherosclerotic cynomolgus monkeys. Topical administration was achieved by injection of adenoviral suspension within the periarterial sheath. Segments of femoral and carotid arteries were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside 1 day after treatment with the adenovirus. After topical administration of virus, beta-galactosidase activity was observed in approximately 20% of cells in the adventitia in both normal and atherosclerotic arteries. There was no detectable beta-galactosidase activity in cells of the intima or media. Thus, topical application provides an alternative method for gene transfer to blood vessels in vivo. This approach, which does not require interruption of blood flow and does not disrupt the endothelium, may be useful for studies of vascular biology and perhaps gene therapy in both normal and atherosclerotic vessels.

Adenovirus-mediated gene transfer in vivo to cerebral blood vessels and perivascular tissue.

Gene transfer to blood vessels in vivo generally requires interruption of blood flow. Thus, gene transduction to cerebral blood vessels in vivo has not yet been achieved. In this study, we injected replication-deficient adenovirus into cerebrospinal fluid in an attempt to transduce genes to cerebral blood vessels. Recombinant adenovirus (1 x 10(9) infectious units) expressing nuclear-targeted bacterial beta-galactosidase driven by the cytomegalovirus promoter was injected into the cisterna magna of Sprague-Dawley rats. The brains were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside 1 to 7 days after injection of adenovirus. Leptomeningeal cells overlying the major arteries were efficiently transduced, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. beta-Galactosidase was expressed on days 1 and 3 after injection but was undetectable by day 7. Expression of the gene was 'targeted' by altering the position of the head. When viral suspension was injected while the rat was in a nose-down position, the reporter gene was expressed extensively on the ventral surface of the brain, especially along the circle of Willis. When the position was changed to the nose-up or lateral position, the inferior or lateral region of the brain was stained primarily. Administration of the virus into the lateral ventricle provided extensive expression in ependymal cells and leptomeninges with some transduction to cerebral blood vessels. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, cisternal delivery may target specific brain regions by positioning of the head.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of sleep-wake habits in older people with changes in output of circadian pacemaker.

Many elderly people complain of disturbed sleep patterns but there is not evidence that the need to sleep decreases with age; it seems rather that the timing and consolidation of sleep change. We tried to find out whether there is a concurrent change in the output of the circadian pacemaker with age. The phase and amplitude of the pacemaker's output were assessed by continuous measurement of the core body temperature during 40 h of sustained wakefulness under constant behavioural and environmental conditions. 27 young men (18-31 years) were compared with 21 older people (65-85 years; 11 men, 10 women); all were healthy and without sleep complaints. The mean amplitude of the endogenous circadian temperature oscillation (ECA) was 40% greater in young men than in the older group. Older men had a lower mean temperature ECA than older women. The minimum of the endogenous phase of the circadian temperature oscillation (ECP) occurred 1 h 52 min earlier in the older than in the young group. Customary bedtimes and waketimes were also earlier in the older group, as was their daily alertness peak. There was a close correlation between habitual waketime and temperature ECP in young men, which may lose precision with age, especially among women. These findings provide evidence for systematic age-related changes in the output of the human circadian pacemaker. We suggest that these changes may underlie the common complaints of sleep disturbance among elderly people. These changes could reflect the observed age-related deterioration of the hypothalamic nuclei that drive mammalian circadian rhythms.

Zidovudine (AZT) reduces virus titer, retards immune dysfunction, and prolongs survival in the LP-BM5 murine induced immunodeficiency model.

Using the murine LP-BM5 retrovirus-induced immunodeficiency model, the therapeutic value of zidovudine (AZT) was analyzed. Continuous low dose (60 mg/kg per day) oral AZT administration for 6 weeks increased survival time by 5-6 weeks. Decreasing the duration of therapy to 3 weeks decreased the mean survival time. Extending the therapy from 6 to 14 weeks increased the median survival time (8 weeks). This dose was nontoxic and reduced virus titers, splenomegaly, and lymphadenopathy. AZT also retarded the immune dysfunction syndrome characteristic of this model. Hypergammaglobulinemia was reduced by AZT and was also a marker for disease progression. AZT reduced hyperproliferation of large blast cells and delayed the loss of splenic B cells.

Bright light resets the human circadian pacemaker independent of the timing of the sleep-wake cycle.

Human circadian rhythms were once thought to be insensitive to light, with synchronization to the 24-hour day accomplished either through social contacts or the sleep-wake schedule. Yet the demonstration of an intensity-dependent neuroendocrine response to bright light has led to renewed consideration of light as a possible synchronizer of the human circadian pacemaker. In a laboratory study, the output of the circadian pacemaker of an elderly woman was monitored before and after exposure to 4 hours of bright light for seven consecutive evenings, and before and after a control study in ordinary room light while her sleep-wake schedule and social contacts remained unchanged. The exposure to bright light in the evening induced a 6-hour delay shift of her circadian pacemaker, as indicated by recordings of body temperature and cortisol secretion. The unexpected magnitude, rapidity, and stability of the shift challenge existing concepts regarding circadian phase-resetting capacity in man and suggest that exposure to bright light can indeed reset the human circadian pacemaker, which controls daily variations in physiologic, behavioral, and cognitive function.