Synergistic effects of p38 mitogen-activated protein kinase inhibition with a corticosteroid in alveolar macrophages from patients with chronic obstructive pulmonary disease.

Corticosteroids partially suppress cytokine production by chronic obstructive pulmonary disease (COPD) alveolar macrophages. p38 mitogen-activated protein kinase (MAPK) inhibitors are a novel class of anti-inflammatory drug. We have studied the effects of combined treatment with a corticosteroid and a p38 MAPK inhibitor on cytokine production by COPD alveolar macrophages, with the aim of investigating dose-sparing and efficacy-enhancing effects. Alveolar macrophages from 10 patients with COPD, six smokers, and six nonsmokers were stimulated with lipopolysaccharide (LPS) after preincubation with five concentrations of dexamethasone alone, five concentrations of the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796) alone, and all combinations of these concentrations. After 24 h, the supernatants were analyzed for interleukin (IL)-8, IL-6, tumor necrosis factor α (TNFα), granulocyte macrophage-colony-stimulating factor (GM-CSF), IL-1α, IL-1ß, IL-1ra, IL-10, monocyte chemoattractant protein 3, macrophage-derived chemokine (MDC), and regulated on activation normal T cell expressed and secreted (RANTES). The effect of dexamethasone on p38 MAPK activation was analyzed by Western blotting. Dexamethasone and BIRB-796 both reduced LPS-induced cytokine production in a dose-dependent manner in all subject groups, with no differences between groups. Increasing the concentration of BIRB-796 in combination with dexamethasone produced progressively greater inhibition of cytokine production than dexamethasone alone. There were significant efficacy-enhancing benefits and synergistic dose-sparing effects (p < 0.05) for the combination treatment for IL-8, IL-6, TNFα, GM-CSF, IL-1ra, IL-10, MDC, and RANTES in one or more subject groups. Dexamethasone had no effect on LPS-induced p38 MAPK activation. We conclude that p38 MAPK activation in alveolar macrophages is corticosteroid-insensitive. Combining a p38 MAPK inhibitor with a corticosteroid synergistically enhances the anti-inflammatory effects on LPS-mediated cytokine production by alveolar macrophages from patients with COPD and controls.

Impaired formation of beta-adrenergic receptor-nucleotide regulatory protein complexes in pseudohypoparathyroidism.

Decreased activity of the guanine nucleotide regulatory protein (N) of the adenylate cyclase system is present in cell membranes of some patients with pseudohypoparathyrodism (PHP-Ia) whereas others have normal activity of N (PHP-Ib). Low N activity in PHP-Ia results in a decrease in hormone (H)-stimulatable adenylate cyclase in various tissues, which might be due to decreased ability to form an agonist-specific high affinity complex composed of H, receptor (R), and N. To test this hypothesis, we compared beta-adrenergic agonist-specific binding properties in erythrocyte membranes from five patients with PHP-Ia (N = 45% of control), five patients with PHP-Ib (N = 97%), and five control subjects. Competition curves that were generated by increasing concentrations of the beta-agonist isoproterenol competing with [125I]pindolol were shallow (slope factors less than 1) and were computer fit to a two-state model with corresponding high and low affinity for the agonist. The agonist competition curves from the PHP-Ia patients were shifted significantly (P less than 0.02) to the right as a result of a significant (P less than 0.01) decrease in the percent of beta-adrenergic receptors in the high affinity state from 64 +/- 22% in PHP-Ib and 56 +/- 5% in controls to 10 +/- 8% in PHP-Ia. The agonist competition curves were computer fit to a "ternary complex" model for the two-step reaction: H + R + N in equilibrium HR + N in equilibrium HRN. The modeling was consistent with a 60% decrease in the functional concentration of N, and was in good agreement with the biochemically determined decrease in erythrocyte N protein activity. These in vitro findings in erythrocytes taken together with the recent observations that in vivo isoproterenol-stimulated adenylate cyclase activity is decreased in patients with PHP (Carlson, H. E., and A. S. Brickman, 1983, J. Clin. Endocrinol. Metab. 56:1323-1326) are consistent with the notion that N is a bifunctional protein interacting with both R and the adenylate cyclase. It may be that in patients with PHP-Ia a single molecular and genetic defect accounts for both decreased HRN formation and decreased adenylate cyclase activity, whereas in PHP-Ib the biochemical lesion(s) appear not to affect HRN complex formation.

Activation of basolateral membrane K+ permeability by bradykinin in MDCK cells.

We study bradykinin-stimulated K+ efflux in Madin-Darby canine kidney (MDCK) cells using 86Rb as an isotopic tracer. Bradykinin brings about a rapid increase in the permeability of MDCK cells to K+, the effect is dose-dependent with a plateau at 10(-6) M. The effect seems to be mediated by Ca2+-activated K+ channels, localised at the basolateral aspect of the epithelium. Unlike alpha-receptors, which mediate a similar effect of adrenalin in these cells, bradykinin receptors seem to be present at both sides of the epithelium. Bradykinin increases the labelling of IP3, and bradykinin-stimulated K+ efflux persists even in cells which are bathed in Ca2+-free medium, suggesting that the effects seen in the present work are probably due to Ca2+ release from intracellular stores. Some extracellular Ca2+ also might be involved in the bradykinin effect, consistent with the kinin-increasing membrane permeability to Ca2+.

Bacterial toxins and the role of ADP-ribosylation.

Studies on the pathogenesis of "Whooping Cough" and cholera have resulted in the discovery of important pathways in the regulation of cellular metabolism leading to the realization of a complex family of proteins that appear to play central roles in the regulation of hormonal responses and which utilize guanine nucleotides in their mechanism of action. The fact that these bacterial toxins interfere so precisely with the complex regulation of eukaryotic cellular metabolism and the discovery of analogous enzymes within the cytosol of eukaryotic cells suggests that ADP-ribosylation may be an important pathway through which the cell can establish its responsiveness to its environment. Clearly, future work directed towards the role of ADP-ribosylation and towards the mechanisms of the regulation of these endogenous ADP-ribosyltransferases and lyases may provide great insights into the mechanisms of hormone action.

Inositol monophosphatase is a highly conserved enzyme having localized structural similarity to both glycerol 3-phosphate dehydrogenase and haemoglobin.

The cDNA coding for an inositol monophosphatase in the oocytes of the African clawed frog, Xenopus laevis, has been isolated and sequenced. The predicted primary structure of this enzyme is markedly conserved when it is compared with its mammalian functional homologues; up to 84% of the amino acid residues are identical, and conservative substitutions increase the similarity to 95%, suggesting that this sequence represents the most parsimonious primary structure for the protein to maintain not only catalytic activity but also perhaps the facility to interact with other macromolecules. Two regions of the protein, each of about 11 residues and separated by about 90 residues, have been identified as a consensus found also in glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). One of these regions is also found to be particularly conserved in the alpha-globin of birds and reptiles; birds and some turtles are known to modulate the oxygen affinity of their haemoglobin with inositol polyphosphate in the same way as with 2,3-bisphosphoglycerate in other species. This region is also conserved in the beta-globin of most species, beginning with lysine-82, which is known to participate in the binding of organic phosphates. These regions of the inositol monophosphatase may represent motifs for the binding of its substrate.

Alkylation of the bovine anterior pituitary D2-dopamine receptor: inconsistency with predictions of the ternary complex model.

The ability of a high affinity, N-chloroethyl derivative to irreversibly occupy dopamine receptors has been used to probe the nature of the interaction of the bovine anterior pituitary D2-dopamine receptor with a putative G-protein. Conditions were developed that resulted in a significant 40-60% decrease in the total D2-dopamine receptor concentration without affecting the affinity of the receptor for either antagonists or agonists. Computer analyses of radioligand binding data for the pituitary D2-dopamine receptor have indicated that consistent with the decrease in total receptor concentration following alkylation there was a comparable decrease in the apparent agonist high-affinity state of the receptor as modelled according to independent classes of binding sites. These data were not consistent with the predictions of the ternary complex model. These results indicate that the present form of the model is not sufficient to account fully for the mechanism of ligand interactions with the pituitary D2-dopamine receptor and suggest, furthermore, that the receptor and the G-protein might be considered as a complex where ligand binding to either component regulates ligand interactions of the other, rather than enhancing the degree of association of the components.

A rapid separation method for inositol phosphates and their isomers.

A technique is described using ACCELL QMA anion-exchange SEP-PAKs (Waters Associates) with ammonium formate-based solutions, whereby a sample can be processed within minutes to yield resolution of inositol phosphates. Isomers of inositol trisphosphate can then be separated by using this technique in combination with a rapid (5-6 min) isocratic h.p.l.c. procedure. The use of QMA SEP-PAKs offers a degree of reproducibility comparable with that of h.p.l.c. while maintaining the capacity for automation, allowing large numbers of samples to be processed rapidly.

Agonist high- and low-affinity states of the D2-dopamine receptor in calf brain. Partial conversion by guanine nucleotide.

In order to determine whether D2-dopaminergic receptors in brain exist in different affinity states for agonists and whether these receptors could be completely converted from their agonist high-affinity state to their agonist low-affinity state, we examined the effect of a guanine nucleotide on the competition between [3H]spiperone and dopamine agonists for binding to homogenates of calf caudate nucleus. [3H]spiperone labeled sites having different affinities for agonists as well as antagonists. Agonists recognized three components of [3H]spiperone binding. Two of these components were related to the D2-dopaminergic receptor. These two sites appeared to represent interconvertible states, each having different affinities for agonists. This was supported by the observation of an apparent guanine nucleotide-induced "conversion" of sites with high affinity to those having low affinity for the agonist. This effect of the guanine nucleotide was incomplete, such that a significant proportion of the high-affinity sites (21%) remained in the presence of an excess of the nucleotide. These high-affinity, guanine nucleotide-insensitive sites may represent a distinct class of binding sites having high affinity for both agonists and antagonists or may be the result at equilibrium of an agonist-independent interaction of the receptor and the guanine nucleotide.

The ternary complex model. Its properties and application to ligand interactions with the D2-dopamine receptor of the anterior pituitary gland.

Agonists and antagonists interact with the pituitary D2-dopamine receptor in a complex fashion that has been accounted for by proposing that the receptor exists in two interconvertible affinity states [De Lean et al., Mol. Pharmacol. 22:290-297 (1982)]. These two states appear to be modulated by guanine nucleotides such that the state existing in the presence of excess guanine nucleotide has low affinity for agonists and high affinity for antagonists. These observations, together with several lines of evidence from other laboratories, have suggested the interaction of the receptor with a guanine nucleotide-binding protein and a model describing the reversible interaction of the receptor (R) with an additional membrane component (X) was studied [De Lean et al., J. Biol. Chem. 255:7108-7117 (1980)]. Several properties of this ternary complex model are presented and discussed in terms of the interpretation of the analysis of simulated binding data using the mass-action model. Computer modeling of experimental binding data obtained from membrane homogenates of bovine anterior pituitary glands indicated that a ternary complex model will fit only under conditions where, in the absence of any ligand, there is a tight interaction or "precoupling" of R with X, with the latter being in stoichiometrically limiting amounts; antagonists and guanine nucleotides would tend to destabilize this interaction, whereas agonists would serve to stabilize the coupled form. These results, for a receptor system that inhibits adenylate cyclase activity, are notably different from those observed for the beta-adrenergic receptor, which stimulates the enzyme and may be a reflection of differences in the molecular mechanisms of the interaction of the two receptor systems with their ligands and their effector. Some features of the model are not compatible with the experimental data and have indicated the need to consider extensions of the model, in light of recent advancements in our understanding of these regulatory components. Our results stress the importance of verifying the properties of proposed models and of cautiously testing these proposed models by their direct application to experimental data.

Automated isocratic high-performance liquid chromatography of inositol phosphate isomers.

Isomers of inositol phosphates from biological samples can be analysed by anion-exchange h.p.l.c., by using isocratic elution with phosphate buffers. The method involves the preliminary processing of the extracted samples with conventional soft-gel anion-exchange resins, including the commonly used Dowex resins, followed by direct analysis with h.p.l.c. of a portion of relevant fractions. Run times (up to 20 min) and collected fraction numbers (up to 24) are minimal, so that if the method is used in conjunction with automated h.p.l.c. injection a high throughput of samples is achieved.

Evidence for receptor-specific regulation of the metabolism of the second messenger, inositol trisphosphate, in human endothelial cells.

Primary cultures (1-2 weeks-old, no passage) of endothelial cells from the human umbilical vein were pre-labelled with [3H]inositol, stimulated for varying times (15 seconds to 5 minutes) in the presence of LiCl with either thrombin or histamine, and the extracted radiolabelled isomers of inositol phosphates were analysed by anion-exchange chromatography. The doses of each receptor-agonist were chosen to stimulate a large accumulation of inositol phosphates at approximately the same rate. At all times of stimulation the relative accumulation of inositol trisphosphate isomers and inositol tetrakisphosphate was different upon stimulation with these agonists, particularly at earlier times. These results argue for the receptor-specific regulation of the inositol trisphosphate 3-kinase. The significance of this observation is discussed with respect not only to the current ideas on the control of calcium homoeostasis by inositol phosphates, but also in consideration of their role as second messengers controlling different end-points of vascular function.

Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors.

Muscarinic receptors were solubilized from porcine atria in digitonin-cholate and were purified by chromatography on DEAE-Sepharose and 3-(2'-aminobenzhydryloxy)tropane-Sepharose. The product identified on Western blots migrated with an apparent molecular mass of 60-75 kDa, with additional bands indicative of homotrimers (190 kDa) and homotetramers (240 kDa). Receptor eluted from the affinity column was accompanied by a mixture of guanyl nucleotide-binding proteins (G-proteins) identified on Western blots as Gi1/2, G(o), Gq/11, and Gs (preparation M2G); the G-proteins were largely removed by further processing on hydroxyapatite (preparation M2). Solubilized purified receptors bound muscarinic ligands in an apparently cooperative manner. In studies at equilibrium, the antagonists [3H]AF-DX 384, N-[3H]methylscopolamine (NMS), and [3H]quinuclidinylbenzilate (QNB) revealed Hill coefficients between about 0.8 and 1.2. Also, the apparent capacity for [3H]QNB exceeded that for [3H]AF-DX 384 and [3H]NMS by about 1.5-fold in M2 and by 2-fold in M2G. Binding to M2G at high concentrations of [3H]QNB was fully inhibited by unlabeled NMS, which therefore affected sites not labeled at similar concentrations of [3H]NMS. Oxotremorine-M displayed a biphasic inhibitory effect on the binding of [3H]AF-DX 384 in M2 and M2G, suggesting that multiple states of affinity are intrinsic to the receptor; 5'-guanylylimidodiphosphate was without appreciable effect in M2 but resulted in a bell-shaped binding profile for the agonist in M2G. All of the data can be described in terms of cooperative interactions within a receptor that is at least tetravalent and presumably an oligomer. In the context of the model, copurifying G-proteins and guanyl nucleotides serve to regulate the degree of cooperativity between successive equivalents of muscarinic ligands.

Inositol phosphate production and Ca2+ mobilization in human umbilical-vein endothelial cells stimulated by thrombin and histamine.

Human umbilical-vein endothelial cells (HUVECs) were cultured, and their inositol phosphate formation and Ca2+ mobilization in response to thrombin and histamine were studied. Evidence from measurement of intracellular Ca2+ in the absence of extracellular Ca2+ established that the two agonists were both acting on a single cell population, and suggested that a Ca2+-influx component was stimulated which was dependent on receptor-occupancy. After 30 s of stimulation in the presence of 10 mM-LiCl, the effects of 20 microM-histamine and 1 unit of thrombin/ml on formation of inositol phosphates were additive, but at 5 min they were not. HUVECs labelled with myo-[3H]inositol for 72 h synthesized radiolabelled inositol pentakis- and hexakis-phosphate. The predominant isomers of inositol mono-, bis- and tris-phosphates whose formation was stimulated were the 4-phosphate, the 1,4-bisphosphate and the 1,3,4-trisphosphate.

Striatal binding of 2-amino-6,7-[3H]dihydroxy-1,2,3,4-tetrahydronaphthalene to two dopaminergic sites distinguished by their low and high affinity for neuroleptics.

In order to develop more selective methods for labeling brain dopamine receptors, this study describes in detail the properties of 2-amino-6,7,-[3H]dihydroxy-1,2,3,4,-tetrahydronaphthalene ([3H] ADTN) binding to dopaminergic sites in rat, calf, and human brain. [3H]ADTN labeled two distinct types of dopaminergic binding sites in the brain striatum of the rat, calf, and human. Very low concentrations of dopamine and dopaminergic catecholamines (with IC50 values of 1 to 10 nM) inhibited the binding of [3H]ADTN to both sites. Neuroleptics, however, inhibited the binding of [3H]ADTN in two distinctly separate concentration ranges, with IC50 values of 0.15 to 40 nM at one site and 100 and 50,000 nM at the other site. The site with high affinity for dopamine and low affinity for neuroleptics had binding properties that corresponded to those of the previously characterized D3 site (List, S., M. Titeler, and P. Seeman (1980) Biochem. Pharmacol. 29: 1621-1622). The [3H]ADTN binding site with high affinity for neuroleptics demonstrated binding characteristics similar to a site labeled by 3H-Neuroleptics (Sokoloff, P., M. P. Martres, and J. C. Schwartz (1980) Naunyn Schmiedebergs Arch. Pharmacol. 315: 89-102). [3H]Apomorphine appeared to label the same two sites as [3H]ADTN, while [3H]dopamine labeled only the D3 site. Scatchard analysis of [3H]ADTN or [3H]apomorphine binding, under conditions for selective labeling of the low affinity neuroleptic site (D3) and the high affinity site for neuroleptics, detected a density of 70 fmol/mg of protein for each. The density of the D3 site in the calf striatum (170 fmol/mg of protein) was much greater than that of the high affinity neuroleptic site (50 fmol/mg). In the rat, the dissociation constant (KD) of [3H]ADTN was 2 nM for both sites. [3H]Apomorphine, however, had a higher affinity for the D3 site (KD=1.6 nM) than for the high affinity neuroleptic site (KD=4.2 nM). The present results may explain previously observed species and laboratory differences between the binding of [3H]ADTN, [3H]apomorphine, and [3H]dopamine.

Dopamine receptor parameters detected by [3H]spiperone depend on tissue concentration: analysis and examples.

The binding of lipophilic radioligands to homogenized tissue was investigated with the help of a simple, two-component model: a specific component reflects binding to a single and uniform population of sites; a nonspecific component reflects partitioning into the membrane and the entrapment of some drug present in the aqueous phase prior to separation of the particulate fraction. The results indicate that the capacity and the affinity of the receptor may be underestimated when the data are analyzed in terms of total rather than free radioligand. Errors in capacity arise when for a significant fraction of the radioligand access to the receptor is blocked by an unlabelled drug and this appears as nonspecific binding. This is most likely to occur when the partition coefficient is such that the free radioligand is located predominantly in the particulate phase. Errors in affinity reflect the tendency of the membrane to reduce the free concentration of a lipophilic drug in the aqueous phase. A further complication arises when a significant fraction of the total radioligand binds to the receptor. [3H]Spiperone binds to dopamine D2 receptors with a dissociation constant of about 50 pM and partitions into the particulate phase of brain homogenates with a membrane/buffer partition coefficient of 410. As expected, both capacity and affinity can appear to depend on the concentration of tissue used in the assay. If the partition coefficient is known, corrected estimates of both parameters can be obtained knowing only the total concentration of radioligand; if the partition coefficient is not known, the free concentration of radioligand in the aqueous phase must be measured independently. The former procedure requires that the aqueous and particulate components of the system be separated by centrifugation; with filtration, the removal of an indeterminate amount of radioligand from the membrane during washing precludes any correction based on the partition coefficient. For the specific example of [3H]spiperone in human brain, the artifacts become negligible at concentrations of protein below 0.1 mg/ml of incubate. The capacity per unit of original tissue is best determined using unwashed preparations, since about 30% of the total protein and a comparable percentage of the receptors are lost on washing.

A mammalian homologue of Drosophila heterochromatin protein 1 (HP1) is a component of constitutive heterochromatin.

The Drosophila HP1 gene contains a highly conserved sequence, the chromobox, which can be used to isolate HP1-like genes from both mouse (M31 and M32) and man (HSM1) (Singh et al., 1991). Here we report that a monoclonal antibody (MoAb) raised against the M31 protein recognises a 26-kDa protein in murine and human nuclear extracts and localises to large masses of condensed chromatin within murine interphase nuclei, some of which are associated with the nucleoli. At metaphase, the MoAb binds to the centromeres of both human and murine chromosomes. The evolutionary conservation of this chromosomal localisation indicates that the M31 protein is likely to be important in the packaging of mammalian chromosomal DNA into constitutive heterochromatin.

Inositol phosphates: proliferation, metabolism and function.

After the initial discovery of receptor-linked generation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) it was generally assumed that Ins(1,4,5)P3 and its proposed breakdown products inositol(1,4)bisphosphate (Ins(1,4)P2) and Ins1P, along with cyclic inositol monophosphate, were the only inositol phosphates found in significant amounts in animal cells. Since then, three levels of complexity have been introduced. Firstly, Ins(1,4,5)P3 can be phosphorylated to Ins(1,3,4,5)P4, and the subsequent metabolism of these two compounds has been found to be intricate and probably different between various tissues. The functions of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are almost certainly to regulate cytosolic Ca2+ concentrations, but the reasons for the labyrinth of the metabolic pathways after their deactivation by a specific 5-phosphatase remain obscure. Secondly, inositol pentakis- and hexakisphosphates have been found in many animal cells other than avian erythrocytes. It has been shown that their synthesis pathway is entirely separate from the inositol phosphates discussed above, both in terms of many of the isomers involved and probably in the subcellular localization; some possible functions of InsP5 and InsP6 are discussed here. Thirdly, cyclic inositol polyphosphates have been reported in stimulated tissues; the evidence for their occurrence in vivo and their possible physiological significance are also discussed.