ABSTRACT
OBJECTIVE: To critically review the pathophysiology of chronic heart failure at the neurohormonal level, and discuss the effect of present and future therapeutic options on these neurohormones. DATA SOURCES: A MEDLINE search (1986-November 2000) was used to identify important primary literature and reviews. Additional references were obtained from these articles. DATA SYNTHESIS: Chronic heart failure is a common, progressive disorder with high morbidity and mortality. Progression is due in large part to several redundant neurohormonal responses. The neurohormones include angiotensin II, norepinephrine, aldosterone, endothelin-1, arginine vasopressin, and tumor necrosis factor. These responses are initially adaptive, but become maladaptive in the long term, impairing the function of the heart, vasculature, and kidneys. Counter-regulatory hormones, such as bradykinin and natriuretic peptides, are insufficient to offset the adverse effects of the other neurohormones. Most drugs used to treat chronic heart failure, such as angiotensin-converting enzyme inhibitors, beta-adrenergic antagonists, and spironolactone, achieve their benefits through altering the neurohormonal pathways. New agents that affect more or different neurohormones may soon be available. CONCLUSIONS: Multiple agents are required for treatment of chronic heart failure, as no single agent can counteract all of the various adverse pathways. The appropriate prescription and use of such inherently complex regimens require significant physician and patient education.
Subject(s)
Cardiac Output, Low/drug therapy , Neurotransmitter Agents/therapeutic use , Chronic Disease , Humans , Kidney/drug effects , Kidney/metabolism , Muscle, Smooth, Vascular/drug effectsABSTRACT
BACKGROUND: Fibromyalgia is a common rheumatologic disorder that is often difficult to treat effectively. CASE SUMMARY: Four patients diagnosed with fibromyalgia syndrome for two to 17 years are described. All had undergone multiple treatment modalities with limited success. All had complete, or nearly complete, resolution of their symptoms within months after eliminating monosodium glutamate (MSG) or MSG plus aspartame from their diet. All patients were women with multiple comorbidities prior to elimination of MSG. All have had recurrence of symptoms whenever MSG is ingested. DISCUSSION: Excitotoxins are molecules, such as MSG and aspartate, that act as excitatory neurotransmitters, and can lead to neurotoxicity when used in excess. We propose that these four patients may represent a subset of fibromyalgia syndrome that is induced or exacerbated by excitotoxins or, alternatively, may comprise an excitotoxin syndrome that is similar to fibromyalgia. We suggest that identification of similar patients and research with larger numbers of patients must be performed before definitive conclusions can be made. CONCLUSIONS: The elimination of MSG and other excitotoxins from the diets of patients with fibromyalgia offers a benign treatment option that has the potential for dramatic results in a subset of patients.
Subject(s)
Feeding Behavior , Fibromyalgia/diet therapy , Fibromyalgia/etiology , Sodium Glutamate/toxicity , Adult , Aspartame/toxicity , Female , Humans , Middle Aged , Neurotoxins/toxicitySubject(s)
Thiazolidinediones , United States Food and Drug Administration/legislation & jurisprudence , Chromans/adverse effects , Cisapride/adverse effects , Cisapride/therapeutic use , Drug and Narcotic Control/legislation & jurisprudence , Humans , Hypoglycemic Agents/adverse effects , Thiazoles/adverse effects , Tobacco Industry/legislation & jurisprudence , Troglitazone , United StatesABSTRACT
We analyzed the endogenous nuclear 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) receptor (VDR) in rat osteosarcoma (ROS 17/2.8) cells and present biochemical evidence that it is a phosphoprotein. When ROS 17/2.8 cells are labeled metabolically with [35S]methionine, treatment with 10(-8) M 1,25(OH)2D3 elicits a decrease in the electrophoretic mobility of immunoprecipitated VDR in denaturing polyacrylamide gels, a property characteristic of phosphorylated proteins. Similar labeling of cells with [32P]orthophosphate results in a rapid (< or = 30 min), 1,25(OH)2D3-dependent incorporation of 32P into a 54-kDa VDR species that comigrates with the slower migrating receptor species extracted from [35S]methionine-labeled ROS 17/2.8 cells that have been exposed to 1,25(OH)2D3. Alkaline phosphatase treatment of immunoprecipitated VDR from 1,25(OH)2D3-treated cells converts the form of the VDR with reduced mobility to the faster migrating species present in 1,25(OH)2D3-deficient cells. Incubation of ROS 17/2.8 cells with the non-hypercalcemic 1,25(OH)2D3 analog, 22-oxacalcitriol (OCT), produces a level of VDR phosphorylation similar to that elicited by 1,25(OH)2D3 treatment. Transient transfection of osteosarcoma cells with a reporter vector containing a vitamin D responsive element derived from the rat osteocalcin gene yields equivalent transcriptional activation in the presence of either 1,25(OH)2D3 or OCT. Further experiments performed at various 1,25(OH)2D3 concentrations to assess the relationship between receptor phosphorylation and transcriptional activity in intact cells showed a positive correlation between these two parameters, indicating that the 1,25(OH)2D3 hormone stimulates VDR phosphorylation and transcriptional activation in parallel. Finally, highly purified casein kinase II (CK-II) phosphorylates the VDR in a 1,25(OH)2D3-independent, in vitro reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Calcitriol/analogs & derivatives , Calcitriol/pharmacology , Osteoblasts/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Steroid/metabolism , Alkaline Phosphatase/metabolism , Animals , Casein Kinase II , Immunosorbent Techniques , Kinetics , Methionine/metabolism , Osteoblasts/drug effects , Osteocalcin/genetics , Osteosarcoma , Phosphates/metabolism , Phosphorylation , Rats , Receptors, Calcitriol , Receptors, Steroid/genetics , Recombinant Proteins/metabolism , Transcription, Genetic , Transfection , Tumor Cells, CulturedABSTRACT
The human 1,25-dihydroxyvitamin D3 receptor (hVDR), like other members of the steroid/thyroid receptor superfamily, has been observed to undergo rapid phosphorylation. We report here that the hVDR is a substrate for casein kinase II (CK-II), a regulatory enzyme of significance in the function of nuclear proteins. Intact hVDRs produced by in vitro transcription/translation or in a baculovirus overexpression system served as efficient substrates for purified bovine CK-II, and the magnitude of this phosphorylation was not affected by the addition of 1,25-dihydroxyvitamin D3. CK-II-catalyzed phosphorylation of truncated hVDRs suggested that phosphorylated residues may occur between Arg121 and Asp232, including the region of hVDR which we have previously demonstrated to contain a major site(s) of phosphorylation in intact cells (Jones, B.B., Jurutka, P.W., Haussler, C.A., Haussler, M.R., and Whitfield, G.K. (1991) Mol. Endocrinol. 5, 1137-1146). Site-directed mutagenesis of serine/threonine residues in this region now reveals a site of phosphorylation at Ser208 contained within the sequence -S208 (P)EEDSDD-, a classic CK-II consensus recognition site. Mutation of this serine to a glycine drastically reduces phosphorylation of hVDR by CK-II, in vitro. The Ser208 mutant receptor also shows a dramatic decrease in [32P]orthophosphate incorporation when transfected into COS-7 cells. We therefore propose that phosphorylation of hVDR at Ser208 in target cells is mediated by casein kinase II or a similar enzyme, and that this quantitatively significant post-translational modification is a potential mechanism for the modulation of the activity of hVDR in controlling gene transcription.
Subject(s)
Calcitriol/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Steroid/metabolism , Serine/metabolism , Amino Acid Sequence , Animals , Casein Kinase II , Cell Line , Chlorocebus aethiops , Humans , Molecular Sequence Data , Moths , Mutation , Phosphorylation , Precipitin Tests , Receptors, Calcitriol , Receptors, Steroid/chemistry , Receptors, Steroid/genetics , TransfectionABSTRACT
The vitamin D receptor (VDR) is known to be a phosphoprotein and inspection of the deduced amino acid sequence of human VDR (hVDR) reveals the conservation of three potential sites of phosphorylation by protein kinase C (PKC)--namely, Ser-51, Ser-119, and Ser-125. Immunoprecipitated extracts derived from a rat osteoblast-like osteosarcoma cell line that contains the VDR in high copy number were incubated with the alpha, beta, and gamma isozymes of PKC, and VDR proved to be an effective substrate for PKC-beta, in vitro. When hVDR cDNAs containing single, double, and triple mutations of Ser-51, Ser-119, and Ser-125 were expressed in CV-1 monkey kidney cells, immunoprecipitated and phosphorylated by PKC-beta, in vitro, the mutation of Ser-51 selectively abolished phosphorylation. Furthermore, when transfected CV-1 cells were treated with phorbol 12-myristate 13-acetate, a PKC activator, phosphorylation of wild-type hVDR was enhanced, whereas that of the Ser-51 mutant hVDR was unaffected. Therefore, Ser-51 is the site of hVDR phosphorylation by PKC, both in vitro and in vivo. To evaluate the functional role of Ser-51 and its potential phosphorylation, hVDR-mediated transcription was tested using cotransfection with expression plasmids and a reporter gene that contained a vitamin D response element. Mutation of Ser-51 markedly inhibited transcriptional activation by the vitamin D hormone, suggesting that phosphorylation of Ser-51 by PKC could play a significant role in vitamin D-dependent transcriptional activation. Therefore, the present results link the PKC signal transduction pathway of growth regulation and tumor promotion to the phosphorylation and function of VDR.
Subject(s)
Mutagenesis, Site-Directed , Protein Kinase C/metabolism , Receptors, Steroid/genetics , Serine , Transcriptional Activation , Amino Acid Sequence , Animals , Brain/enzymology , Calcitriol/metabolism , Cell Line , Cloning, Molecular , Escherichia coli/genetics , Genetic Vectors , Humans , Mice , Phosphorylation , Protein Kinase C/isolation & purification , Receptors, Calcitriol , Receptors, Steroid/metabolism , Recombinant Proteins/metabolism , Transcription, Genetic , TransfectionABSTRACT
A baculovirus expression vector system (BEVS) was used to overproduce the full-length human vitamin D receptor (hVDR) in Spodoptera frugiperda ovarian cells. hVDR was expressed to a level of 0.5% of the total soluble protein in this system. Western analysis demonstrated that the baculovirus-generated protein had electrophoretic and immunologic properties equivalent to those of hVDR expressed in mammalian cells. The BEVS-derived receptor displayed specificity and high affinity (apparent Kd = 0.7 nM) for the 1,25(OH)2D3 ligand. Recombinant hVDR generated a specific protein-DNA complex with a duplex oligomer containing a vitamin D-responsive element (VDRE) in gel mobility shift assays. The intensity of the VDR.VDRE complex was not affected by 1,25(OH)2D3. However, the complex exhibited increased mobility in the presence of hormone, possibly the result of a 1,25(OH)2D3-dependent conformational change. A nuclear extract obtained from CV-1 cells markedly enhanced the intensity of this VDR.VDRE complex and produced an additional distinct VDR-dependent complex, thus implicating a role for nuclear auxiliary factors in multiple high affinity VDR.VDRE interactions. Finally, methylation interference studies defined the guanine residues contacted when the putative VDR-auxiliary factor complex associates with the rat osteocalcin VDRE; specifically, all of the GC base pairs in the sequence GGGTGAATGAGGACA. Therefore, these results show that the BEV system elicits high level expression of hVDR with critical functional characteristics being preserved.
Subject(s)
Baculoviridae/genetics , Receptors, Steroid/genetics , Vitamin D/metabolism , Animals , Base Sequence , Blotting, Western , Cloning, Molecular , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genetic Vectors , Humans , Methylation , Molecular Sequence Data , Osteocalcin/genetics , Osteocalcin/metabolism , Rats , Receptors, Calcitriol , Receptors, Steroid/metabolismABSTRACT
The gene for rat bone gla protein (BGP) was isolated and 1250 basepairs (bp), including 1100 bp of 5' flanking DNA, were placed up-stream of the human GH reporter gene. After transient transfection into the osteoblast-like rat osteosarcoma cell line ROS 17/2.8, the BGP promoter demonstrated a low level of basal activity that was increased approximately 10-fold by the addition of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. A single 250-bp fragment (-523 to -274) was sufficient to confer hormone inducibility upon both heterologous and homologous promoters. Deletion studies, complemented by evaluation with synthetic oligomers, enabled localization of the 1,25-(OH)2D3 response element to within 19 bp (-456 to -438), containing an element with an imperfect direct repeat [GGTGA(N4)GGACA] and homology to other steroid-responsive elements. Gel retardation assays demonstrated that partially purified chick intestinal 1,25-(OH)2D3 receptor bound specifically and with high affinity to a DNA fragment containing the putative 1,25-(OH)2D3 response element, and this binding was perturbed by monoclonal antibodies to the 1,25-(OH)2D3 receptor. Surprisingly, the 250-bp fragment, when linked in an antisense orientation with respect to the BGP promoter, blocked basal and hormone-dependent gene expression. However, a 246-bp fragment 5' to the 250-bp element (-1100 to -855) restored 20-fold inducibility when linked to the first fragment in the same orientation, suggesting cooperativity between at least two elements to achieve the hormonal regulation observed in this gene.
Subject(s)
Bone and Bones/metabolism , Calcitriol/pharmacology , Calcium-Binding Proteins/genetics , Enhancer Elements, Genetic , Extracellular Matrix Proteins , Gene Expression Regulation , Repetitive Sequences, Nucleic Acid , Animals , Base Sequence , Bone and Bones/drug effects , Chickens , DNA/chemistry , Humans , Intestines/chemistry , Models, Molecular , Molecular Sequence Data , Rats , Receptors, Calcitriol , Receptors, Steroid/genetics , Restriction Mapping , Matrix Gla ProteinABSTRACT
A fragment of the complementary deoxyribonucleic acid to the human 1,25-dihydroxyvitamin D3 receptor protein containing essentially the entire open reading frame was transcribed and translated in vitro. The resulting protein was then demonstrated to exhibit the physical and functional features, i.e. molecular weight, immunoreactivity, 1,25-dihydroxyvitamin D3 binding, and DNA-cellulose binding, of the native human receptor from the T47D cell line. This validates the authenticity of the cDNA in a cell free system and provides a biochemical means of generating this rare and labile macromolecule to use in heretofore difficult structure/function studies.
Subject(s)
Cell-Free System/metabolism , DNA/biosynthesis , Receptors, Steroid/genetics , Calcitriol/metabolism , Cell Line , Humans , In Vitro Techniques , Molecular Weight , Open Reading Frames , Precipitin Tests , Protein Biosynthesis , Receptors, Calcitriol , Receptors, Steroid/biosynthesis , Structure-Activity Relationship , Transcription, GeneticABSTRACT
High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.