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Myron L Toews

Publications and source records attributed to Myron L Toews.

12 recordsLinked to original sources
PubMed2006

Adenosine receptors find a new partner and move out.

Recent studies of G protein-coupled receptors have highlighted two "new" and interactive elements involved in their function and regulation: their ability to localize to different cell surface and intracellular compartments and their ability to interact with partners other than their classic heterotrimeric G proteins. The effects mediated by these receptors can be markedly different depending on the compartment in which they reside and the partners with which they interact in each compartment. The studies in this issue of Molecular Pharmacology by Milojević et al. (page 1083) merge these two themes by identifying the ubiquitin-specific protease Usp4 as a partner for the carboxyl-terminal tail of adenosine A2A receptors and showing that this interaction allows processing and delivery to the cell surface of newly synthesized A2A receptors, which are otherwise predominantly intracellular. Their studies suggest that the intracellular A2A receptors are ubiquitinated, presumably because of misfolding and intervention of the "endoplasmic reticulum quality control" mechanism leading to degradation of the receptors by proteasomes. Increasing Usp4 expression stimulates receptor de-ubiquitination and increases cell surface expression of functional receptors. Evidence is presented for tight specificity of this interaction, with another Usp family member failing to rescue A2A receptors and Usp4 failing to rescue another intracellular receptor. The background and significance of this study are highlighted here, including puzzles that remain to be solved and the potential for pharmacological targeting of such interactions to manipulate the expression, location, and function of G protein-coupled receptors for therapeutic benefit.

Humans↗
PubMed2005

Pharmacologic principles for combination therapy.

This article discusses the pharmacologic basis for understanding the therapeutic actions of drugs, particularly for their use in combinations. The focus is on principles underlying combination therapy in general, including examples from diseases other than chronic obstructive pulmonary disease (COPD). Pharmacodynamic aspects of drug action are covered, with an emphasis on recent advances in the understanding of drug-receptor interactions and of drug agonism. Pharmacokinetics and drug-induced adaptive changes in receptors and cell signaling pathways are summarized, emphasizing their importance for potential combination therapies aimed at prolonging drug action. An organizational framework for three different approaches to combination therapy is then proposed; the molecular rationales for each approach are described together with classic examples from other diseases, and then their application to combination therapy in COPD is discussed. Finally, terminology for the independent and interactive effects of drug combinations is discussed, and approaches to the quantitative analysis and visual display of the effects of drug combinations are introduced. The basic principles reviewed here provide the pharmacologic foundation for subsequent articles in this issue that address the combinations in current use for COPD, and they point to novel strategies for potential future approaches to combination therapy in COPD.

Drug Interactions↗
PubMed2004

The N terminus of the human alpha1D-adrenergic receptor prevents cell surface expression.

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A)NT-D, alpha(1B)NT-D, alpha(1D)NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)-or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

Binding Sites↗
PubMed2004

Radioligand-binding methods for membrane preparations and intact cells.

The radioligand-binding assay is a relatively simple but powerful tool for studying G-protein-coupled receptors. There are three basic types of radioligand-binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the binding site density can be determined; (2) inhibition experiments from which the affinity of a competing, unlabeled compound for the receptor can be determined: and (3) kinetic experiments from which the forward and reverse rate constants for radioligand binding can be determined. Detailed methods for typical radioligand-binding assays for G-protein-coupled receptors in membranes and intact cells are presented for these types of experiments. Detailed procedures for analysis of the data obtained from these experiments are also given.

Animals↗
PubMed2003

Regulation of alpha-1B adrenergic receptor localization, trafficking, function, and stability.

The alpha-1 adrenergic receptors (alpha(1)ARs) play important roles in normal physiology and in many disease states, and understanding their signaling pathways and regulatory mechanisms is thus of considerable relevance, in particular for identifying pharmacological targets for therapeutic modulation. The expression, function, localization, trafficking, and stability of these receptors are all subject to complex regulation by diverse molecular mechanisms. This article highlights recent studies from our laboratory and others focused on the localization and trafficking of the alpha-1B adrenergic receptor (alpha(1B)AR) subtype and on changes in its stability that are likely to be involved in regulating receptor expression. The role(s) of protein kinase C in alpha(1B)AR sequestration, endocytosis, and extracellular signal-regulated kinase (ERK) activation are summarized, and evidence for alpha(1B)AR localization in caveolae/rafts is presented. Receptor structural domains involved in the multiple steps and mechanisms of agonist-induced desensitization are described. Finally, aspects of alpha(1B)AR structural stability that appear to control its drug-induced up- and down-regulation are discussed. Our understanding of regulation for the alpha(1B)AR subtype provides a model for studies of the differential regulation of the other alpha(1)AR subtypes and may lead to identification of new molecular targets for therapeutic intervention in a variety of disease states.

Animals↗
PubMed2003

Up-regulation of alpha1B-adrenergic receptors with defects in G protein coupling: ligand-induced protection from receptor instability.

The biochemical basis for the unexpected agonist-induced up-regulation of the number of radioligand binding sites for two mutated alpha1B-adrenergic receptors reported previously was investigated. Up-regulation was independent of the expression vector used and was not prevented by cycloheximide or actinomycin D, eliminating several potential transcriptional mechanisms and new receptor protein synthesis. Antagonists were also able to induce up-regulation, suggesting that ligand occupancy without signal generation was sufficient to induce the increase in binding sites. Accordingly, we hypothesized that up-regulation results from ligand-induced protection from inherent instability of these mutated receptors. Studies with receptors in isolated membranes revealed that the two mutated receptors that exhibited up-regulation in intact cells also exhibited an inherent instability of their ligand binding capacity, and binding of either agonists or antagonists to these receptors could protect against the loss of binding. In contrast, the wild-type receptor and other mutated receptors that did not exhibit up-regulation in intact cells did not exhibit instability or ligand-induced protection in isolated membranes. The occurrence of instability and protection in isolated membranes for only those mutated receptors and ligands that exhibit up-regulation in intact cells provides compelling evidence that the apparent up-regulation of binding sites in intact cells results from ligand-induced protection from an inherent instability of these G protein coupling-defective receptors. Inclusion of protease inhibitors markedly reduced the loss of binding in isolated membranes, implicating membrane-localized proteolysis as the likely mechanism for the instability.

Adrenergic alpha-Agonists↗
PubMed2002

Lysophosphatidic acid in airway function and disease.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator and important component of serum. Studies over the past several years which have documented diverse effects of LPA on multiple types of airway cells and which suggest possible involvement of LPA in lung disease are reviewed here. LPA enhances contractility of airway smooth muscle. It also stimulates proliferation of cultured airway smooth muscle cells and exhibits a striking synergism with epidermal growth factor (EGF) for stimulating mitogenesis. Recent studies of the molecular components and signaling pathways mediating synergism are described, including LPA-induced upregulation of EGF receptors and activation of multiple transcription factors by both LPA and EGF. A model for the effects of LPA and EGF on mitogenesis that includes EGF receptor upregulation and synergism between Ras and Rho for activation of the transcription factor AP-1 is presented. LPA stimulates fibronectin secretion and filopodia extension in airway epithelial cells as well as proliferation and collagen gel contraction by lung fibroblasts. A hypothesis for LPA involvement in the airway repair and remodeling, which contribute to the pathology of asthma and other airway diseases, is presented, and future directions for research into the roles of LPA in airway function and disease are suggested.

Animals↗
PubMed2002

Lysophosphatidic acid augments fibroblast-mediated contraction of released collagen gels.

Lysophosphatidic acid (LPA) is a glycerophospholipid released from platelets that has multiple biologic effects. The present study evaluated the potential of LPA to modulate tissue repair and remodeling by modifying human lung fibro-blast-mediated contraction of three-dimensional collagen gels. The contraction of native collagen gels caused by human fetal lung fibroblasts was augmented by LPA in a concentration-dependent manner. The estimated median effective concentration was 3 x 10(-7) mol/L, which was well below the concentrations likely released by platelets in tissues. LPA-augmented contraction was not blocked by pertussis toxin or cholera toxin but was inhibited by inhibition of phospholipase C. Neither calcium mobilization nor protein kinase C appeared to play a role. In contrast, the effect of LPA appeared to depend on a kinase inhibited by staurosporine but not by genistein or GF109203X, suggesting a process that depends on phospholipase C and may involve a novel protein kinase. By modulating fibroblast-mediated remodeling, LPA could play a role in the tissue remodeling that characterizes wound repair.

Calcium↗
PubMed2002

Regulatory properties of alpha(1B)-adrenergic receptors defective in coupling to phosphoinositide hydrolysis.

Previous studies have suggested that G protein coupling, phospholipase C activation, phosphoinositide hydrolysis, and protein kinase C activation may be required for alpha(1B)-adrenergic receptor regulation, particularly for their endocytosis into intracellular vesicles. Accordingly, the internalization and down-regulation properties of mutated receptors with defects in G protein coupling and second messenger generation were investigated. The Delta12 and Delta5 receptors, previously shown to be defective in G protein coupling, exhibited greater agonist-induced losses of cell surface accessibility assessed by radioligand binding to intact cells on ice than for the wild-type receptor; however, these receptors were completely defective in endocytosis into intracellular vesicles assessed by sucrose density gradient centrifugation. These receptors also did not undergo down-regulation with long-term agonist exposure as did the wild-type receptor; instead, a prominent up-regulation was observed. The Y348A receptor, previously shown to be defective in phosphoinositide hydrolysis and endocytosis was also defective in down-regulation but did not exhibit significant up-regulation. In contrast, a receptor construct with amino acid residues 246 to 261 deleted (Delta[246-261]) was also defective in stimulation of phosphoinositide hydrolysis but exhibited internalization and down-regulation properties essentially identical to those for the wild-type receptor. Together, these results suggest that stimulation of phosphoinositide hydrolysis by alpha(1B)-adrenergic receptors is not required for their endocytosis or down-regulation but that similar and overlapping receptor structural domains are involved in mediating these processes.

Adrenergic beta-Agonists↗
PubMed2002

Lysophosphatidic acid upregulates the epidermal growth factor receptor in human airway smooth muscle cells.

Human airway smooth muscle cells treated with lysophosphatidic acid (LPA) and epidermal growth factor (EGF) exhibit synergistic stimulation of mitogenesis (Ediger TL and Toews ML. J Pharmacol Exp Ther 294: 1076-1082, 2000). The effects of LPA treatment of human airway smooth muscle cells on EGF receptor (EGFR) regulation have now been investigated. LPA treatment for 12-24 h resulted in a twofold increase in (125)I-EGF binding and EGFR protein levels as assessed by Western blot analysis. Competition binding assays indicated single-site binding with an affinity of 3 nM, and the affinity was not changed by LPA treatment. EGFR upregulation was blocked by cycloheximide and actinomycin D, suggesting that LPA influences transcriptional regulation of EGFR expression. Inhibitor studies revealed a prominent role for activation of mitogen-activated protein kinase and p70 ribosomal S6 kinase. Both synergism and EGFR upregulation increased with increased cell density, whereas EGFR expression in control cells decreased. The similar requirements for exposure time, LPA concentrations, and cell confluence suggest that EGFR upregulation may be one contributing factor to the synergistic stimulation of mitogenesis seen with LPA plus EGF.

Binding, Competitive↗
PubMed2002

Lysophosphatidic acid receptor signaling in mammalian retinal pigment epithelial cells.

PURPOSE: Lysophosphatidic acid (LPA) is a phospholipid growth factor that stimulates proliferation, chemotaxis, cation currents, and K(+) currents in retinal pigment epithelial (RPE) cells. LPA receptor transduction was analyzed in human and rat RPE cells. METHODS: Cells were cultured with standard methods, and signaling pathways were analyzed with a variety of approaches, including whole-cell recording, calcium imaging, and second-messenger assays. RESULTS: LPA-activated nonselective cation currents in rat RPE were blocked by the protein tyrosine kinase (PTK) inhibitor genistein, by the MAP kinase kinase (MEK) inhibitor PD98059, and by loading cells with antibodies to G(alpha(i)/o/t/z). LPA activated the MAP kinase and extracellular signal-related kinase (ERK)-1, and produced a dose-dependent inhibition of cAMP production. LPA stimulated a dose-dependent increase in [Ca(2+)](i) that persisted in Ca(2+)-free medium and was reduced by pretreatment with thapsigargin, suggesting it involves release from intracellular stores. The [Ca(2+)](i) increase was not blocked by ryanodine or the phospholipase C inhibitor U73122. LPA did not stimulate inositol phosphate production. Similar to the cation current, LPA-evoked [Ca(2+)](i) increases were blocked by PD98059 and by loading cells with antibodies to G(alpha(i)/o/t/z). RT-PCR experiments showed the presence of RNA for three LPA receptor subtypes (Edg2, -4, and -7); RNase protection assays showed the strongest expression for Edg2 receptor RNA. CONCLUSIONS: LPA receptors in RPE cells activate pertussis toxin (PTx)-sensitive G proteins that inhibit cAMP accumulation; stimulate MAP kinase which activates a cation current and probably contributes to mitogenesis; and stimulate release of Ca(2+) from intracellular stores that appears independent of IP(3) and ryanodine receptor activation.

Animals↗