Analysis of wear, wear particles, and reduced inflammatory potential of vitamin E ultrahigh-molecular-weight polyethylene for use in total joint replacement.

Vitamin E (VE) has been added to ultrahigh-molecular-weight polyethylene (UHMWPE) acetabular cups and tibial trays primarily to reduce oxidative damage to the polymer. The aim of this study was to investigate the relative wear rates of UHMWPE-containing VE compared with virgin UHMWPE. The ability of VE to reduce the amount of inflammatory cytokines produced from stimulated peripheral blood mononuclear cells (PBMNCs) was also investigated. Stimulation was achieved by exposure of PBMNCs to either lipoplysaccharide (LPS) or VE-containing UHMWPE (VE-UHMWPE). In the present study, results showed that the wear rates of UHMWPE with or without VE were not significantly different. Particles generated by UHMWPE with and without VE were not significantly different in size distribution. The production of osteolytic mediators, tumor necrosis factor-alpha, interleukin 1ß (IL-ß), IL-6, and IL-8 were significantly reduced in (PBMNCs) stimulated with either LPS + VE compared with LPS or VE-UHMWPE particles compared to virgin UHMWPE particles. This trend was also observed when VE was added as a liquid to UHMWPE wear particle-stimulated PBMNCs. The exact mechanism of how VE affects the release of inflammatory mediators from particle-stimulated macrophages is not yet understood. It is likely to involve the anti-inflammatory and/or antioxidant effects of VE.

In vitro analysis of the cytotoxic and anti-inflammatory effects of antioxidant compounds used as additives in ultra high-molecular weight polyethylene in total joint replacement components.

Ultra high-molecular weight polyethylene (UHMWPE) remains the most commonly used material in modern joint replacement prostheses. However, UHMWPE wear particles, formed as the bearing articulates, are one of the main factors leading to joint replacement failure via the induction of osteolysis and subsequent aseptic loosening. Previous studies have shown that the addition of antioxidants such as vitamin E to UHMWPE can improve wear resistance of the polymer and reduce oxidative fatigue. However, little is known regarding the biological consequences of such antioxidant chemicals. This study investigated the cytotoxic and anti-inflammatory effects of a variety of antioxidant compounds currently being tested experimentally for use in hip and knee prostheses, including nitroxides, hindered phenols, and lanthanides on U937 human histocyte cells and human peripheral blood mononuclear cells (PBMNCs) in vitro. After addition of the compounds, cell viability was determined by dose response cytotoxicity studies. Anti-inflammatory effects were determined by quantitation of TNF-α release in lipopolysaccharide (LPS)-stimulated cells. This study has shown that many of these compounds were cytotoxic to U937 cells and PBMNCs, at relatively low concentrations (micromolar), specifically the hindered phenol 3,5-di-tert-butyl-4-hydroxyhydrocinnamate (HPAO1), and the nitroxide 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMPO). Lanthanides were only cytotoxic at very high concentrations and were well tolerated by the cells at lower concentrations. Cytotoxic compounds also showed reduced anti-inflammatory effects, particularly in PBMNCs. Careful consideration should therefore be given to the use of any of these compounds as potential additives to UHMWPE.

Molecular basis of protein kinase C-induced activation of ATP-sensitive potassium channels.

Potassium channels that are inhibited by internal ATP (K(ATP) channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K(ATP) channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K(ATP) channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC similarly regulates recombinant channels from both the pancreas and heart. Surprisingly, PKC acts via phosphorylation of a specific, conserved threonine residue (T180) in the pore-forming subunit (Kir6.2). Additional PKC consensus sites exist on both Kir and the larger sulfonylurea receptor (SUR) subunits. Nonetheless, T180 controls changes in open probability induced by direct PKC action either in the absence of, or in complex with, the accessory SUR1 (pancreatic) or SUR2A (cardiac) subunits. The high degree of conservation of this site among different K(ATP) channel isoforms suggests that this pathway may have wide significance for the physiological regulation of K(ATP) channels in various tissues and organelles.

Nitric oxide regulates cyclic GMP-dependent protein kinase phosphorylation in rat brain.

Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.

Autoradiographic localization of [3H]-cyclic GMP binding sites in the rat brain.

Both the atriopeptides and nitric oxide act in the nervous system by activating guanylyl cyclases to stimulate the production of cyclic GMP. Thus a key to understanding the roles of these messengers is to understand the functions of cyclic GMP in the nervous system. Three potential targets for cyclic GMP have been identified, phosphodiesterases, protein kinases and ion channels. In this study we describe a method using autoradiography to localize specific [3H]-cGMP binding sites in the brain. The specific binding of [3H]-cGMP to rat brain sections was saturable (Bmax = 1.5 pmol/mg protein) and of high affinity (KD = 164 nM). The pharmacological characteristics were consistent with binding to the cGMP-dependent protein kinase. Highest densities of binding were seen in the medial habenula, basal ganglia, locus ceruleus and nucleus of the solitary tract. The CA1 pyramidal cells of the hippocampus, the neocortex, thalamus and cerebellum were also labelled. This method should prove useful in studies of potential targets for cyclic GMP in the brain.

Expression of the olfactory cyclic nucleotide gated channel (CNG1) in the rat brain.

The expression of the olfactory cyclic nucleotide-gated channel (CNG1) was studied in the rat brain. Using RT-PCR, levels of CNG1 mRNA were determined relative to the expression of a constantly expressed gene, alpha-tubulin. RT-PCR showed that CNG1 mRNA was detectable in the pituitary gland, the olfactory bulb, and the cerebellum of adult and 5-day-old rats. A 3.4 kb mRNA was detected in the olfactory bulb by Northern blotting. In situ hybridization analysis showed that CNG1 mRNA expression is present in the olfactory bulb and in the Purkinje cells of the cerebellum. RT-PCR studies on Purkinje cell-enriched cultures obtained from the cerebellum of 16-day-old embryos (E16) confirmed the expression of CNG1 mRNA in these neurones. Our results show that CNG1 is not restricted to the olfactory epithelium but is also present in specific regions of the brain. These results suggest that cyclic nucleotides may act in the regions that possess CNG1 gene expression to affect the electrical activity of certain neurones directly.

Molecular characterization of a type II cyclic GMP-dependent protein kinase expressed in the rat brain.

We applied reverse transcription-PCR to examine the gene expression of cyclic GMP (cGMP)-dependent protein kinase in the rat brain. A PCR product with the size predicted from the type II cGMP-dependent protein kinase (cGK II) cDNA was detected in various regions of the brain, with highest expression in the thalamus. The amplified product of this cDNA was subcloned, sequenced, and consequently shown to be cGK II. Northern analysis confirmed that this kinase was highly expressed in the thalamus. In situ hybridization with riboprobes derived from this cDNA indicated that cGK II mRNA was highly expressed in the outer layers of the cortex, the septum, amygdala, and olfactory bulb with highest levels in the thalamus. High amounts of cGK II mRNA were also found in specific brainstem loci, including the medial habenula, the subthalamic nucleus, the locus ceruleus, the pontine nucleus, the inferior olivary nuclei, and the nucleus of the solitary tract. Only low levels of cGK II mRNA were detected in the striatum, cerebellum, and hippocampus. These data suggest that the effects of guanylyl cyclase activators, such as nitric oxide and the atriopeptides, in various regions of the CNS may be mediated through cGK II.

Characterization and localization of [3H] cyclosporin A binding sites in rat brain.

The immunosuppressant drug [3H]cyclosporin A binds specifically and with high affinity to rat brain membrane preparations. The highest density of binding sites was observed in the hippocampus, cerebellum, cortex and basal ganglia. A similar distribution pattern was seen using a quantitative autoradiographic analysis. This distribution agrees with the localizations of cyclophilin and calcineurin reported in immunohistochemical and in situ hybridization studies. Thus inhibition of calcineurin activity following cyclosporin A binding to cyclophilin may occur in neurones, as it does in T-cells. These results suggest that the neurological side-effects of cyclosporin A may be mediated through its interaction with these proteins in neurones.

Low-affinity binding sites for 1,4-dihydropyridines in skeletal muscle transverse tubule membranes revealed by changes in the fluorescence of felodipine.

The fluorescence changes accompanying the binding of the fluorescent calcium channel antagonist, felodipine, to transverse tubule membranes from rabbit skeletal muscle have been used to characterize low-affinity binding sites for 1,4-dihydropyridine derivatives in these preparations. In competition experiments, felodipine inhibited the high-affinity binding of (+)-[3H]PN200-110 to transverse tubule membranes with an apparent Ki of 5 +/- 2 nM. Binding of felodipine to additional low-affinity sites resulted in a large, saturable (Kd = 6 +/- 2 microM) increase in its fluorescence which could be excited either directly (380 nm) or indirectly via energy transfer from membrane protein (290 nm). The observed fluorescence enhancement was competitively inhibited by other 1,4-dihydropyridines with inhibition constants of 3-21 microM but was unaffected by the structurally unrelated calcium channel antagonists, diltiazem and verapamil, or by Ca2+, Cd2+, and La3+. Both high- and low-affinity binding sites appear to be localized in the transverse tubular system, since the magnitude of the observed fluorescence enhancement was higher in these membranes than in microsomal preparations and was directly proportional to the density of high-affinity sites for (+)-[3H]PN200-110. Furthermore, both high- and low-affinity sites appear to be conformationally coupled since, over the same concentration range that the fluorescence changes were observed, felodipine accelerated the rate of dissociation of [3H]PN200-110 previously bound to its high-affinity sites. Similar behavior has previously been reported for other 1,4-dihydropyridines [Dunn, S. M. J., & Bladen, C. (1991) Biochemistry 30, 5716-5721].(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: evidence for low-affinity sites and for the involvement of G proteins.

Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110 to membrane preparations from rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites (Kd = 0.30 +/- 0.05 nM) that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]-PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 microM for the agonist (+/-)-Bay K8644 and for the antagonists nifedipine, (+/-)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since (-)-PN200-110 (1-200 microM) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) on binding parameters. At a concentration of 10 microM, neither GTP gamma S nor GDP beta S significantly affected the binding of dihydropyridines to their high-affinity sites. GTP gamma S did, however, increase the ability of (+/-)-Bay K8644, but not of (+/-)-nitrendipine, to accelerate the rate of dissociation of tightly bound (+)-[3H]PN200-110. GDP beta S did not affect the dose dependence of either the agonist or the antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypertension and the Department of Health and Human Services.

Hypertension affects from 17% to 25% of all Americans. Because of its fundamental charge to help protect the public health, the Department of Health and Human Services (HHS) is substantially concerned with the condition. Additionally, hypertension represents a significant source of underwriting risk to which HHS is exposed in its role as health insurer of the poor and the elderly. HHS has invested hundreds of millions of dollars in hypertension research, development and testing of treatment regimens, and education of health care providers and consumers. However, much of the etiology of hypertension still eludes us. Sodium's apparent importance as a "causal" agent to the development of hypertension, and in its treatment, has waxed and waned over the past several generations; research to date has not yet finally settled the issue. Is sodium or some other cation the key? While research on this issue continues, HHS is currently faced with deciding whether and how to require inclusion of sodium content in nutrition labelling. In the debate, attention has to be given not only to the current best evidence on sodium; additional issues of consumer choice, costs, and education are also of importance.