Biomedical subjects
R M Edwards
Publications and source records attributed to R M Edwards.
alpha-Ketoglutarate transport in rat renal brush-border and basolateral membrane vesicles.
The dicarboxylate, alpha-ketoglutarate (alphaKG), has been identified as the most likely physiological anion involved in renal proximal tubule basolateral membrane (BLM) dicarboxylate/organic anion exchange. In the present study, we characterized the uptake of alphaKG in BLM and brush-border membrane (BBM) vesicles isolated from rat kidney. In both membrane preparations, alphaKG uptake was Na+-dependent, saturable, electrogenic and inhibited by Li+. The initial rate of alphaKG (5 microM) uptake in BLM vesicles was twice that in BBM vesicles (258 +/- 8.2 vs. 126 +/- 3.9 pmol/mg/5 sec). The BLM transporter had a high affinity for alphaKG (apparent Km = 15.2 microM), but a relatively low transport capacity (Vmax = 386 pmol/mg/5 sec). In contrast, the BBM transporter had characteristics of a low-affinity (Km = 158 microM), high-capacity (Vmax = 1106 pmol/mg/5 sec) system. Other dicarboxylates such as succinate, malate, fumarate and glutarate at a concentration of 1 mM inhibited alphaKG uptake into BLM and BBM vesicles to the same extent (>90%). The tricarboxylate, citrate, also inhibited alphaKG uptake (70-80%). However, of these Krebs' cycle intermediates, only alphaKG and glutarate were able to affect p-aminohippurate (PAH) uptake into BLM vesicles. These results lend further support for a BLM PAH/alphaKG exchanger. Furthermore, if extracellular alphaKG plays a role in the operation of the PAH/alphaKG exchanger, the high-affinity Na+-dependent alphaKG transporter located in the BLM is the likely source of the organic anion.
Phosphate excretion and phosphate transporter messenger RNA in uremic rats treated with phosphonoformic acid.
The prevention of phosphate retention in chronic renal disease may reduce both renal osteodystrophy and disease progression. We evaluated the expression of the sodium-dependent phosphate transporter, NaPi-2, and the response to phosphonoformic acid (PFA) in rats with 5/6 nephrectomy-induced renal failure. Partial nephrectomy resulted in a significant proteinuria and reduced renal function. In addition, there was an approximately 50% reduction in the expression of NaPi-2 mRNA. Treatment of rats for 48 hr with PFA (0.6% in glucose drinking fluid) had no effect on NaPi-2 mRNA; however, PFA resulted in a significant increase in fractional phosphate excretion in both normal (7 +/- 0.5% vs. 3 +/- 0.2%) and uremic (60 +/- 4% vs. 36 +/- 4%) rats. Plasma phosphate concentration was higher in uremic rats (2.5 +/- 0.1 mM) compared with normal rats (1.9 +/- 0.04 mM) but not in uremic rats treated with PFA (2.1 +/- 0.04 mM). These data suggest that PFA can increase renal phosphate excretion independent of changes in phosphate transporter expression and prevent phosphate retention.
Evaluation of the Roche Amplicor polymerase chain reaction system for detection of Mycobacterium tuberculosis complex in specimens.
Roche Diagnostic Systems Inc. have recently developed a commercial PCR (Amplicor) for direct amplification of Mycobacterium tuberculosis Complex (MtbC) from sputum and bronchial washes. Detection of MtbC specific sequence is achieved by hybridization with an oligonucleotide probe. The aim of this study was to compare the Amplicor PCR system for detection of MtbC with microscopy, culture and an in-house PCR method. The commercial assay correctly identified 35/37 microscopy positive specimens compared to 34/37 with the in-house method and 7/13 microscopy negative, culture positive samples compared to 2/13 with the in-house procedure. Negative PCR results were obtained for 32 culture negative specimens and 6 specimens which yielded mycobacteria other than MtbC, indicating a specificity of 100%. The sensitivity of the commercial assay was determined to be approximately 10 organisms compared to 100 organisms with the in-house method. The Amplicor PCR system is specific, sensitive and easy to perform. It also has the advantages of being standardized and quality controlled.
Effect of adrenomedullin on cAMP levels along the rat nephron: comparison with CGRP.
Adrenomedullin (ADM) is a recently discovered hypotensive peptide that has been detected in the kidney. Recent studies have shown that intrarenal administration of ADM produces natriuresis without changes in glomerular filtration rate. Although ADM stimulates adenosine 3',5'-cyclic monophosphate (cAMP) synthesis in some tissues, the site of action and signal transduction pathway in the kidney are unknown. In the present study, we examined the effects of ADM and calcitonin gene-related peptide (CGRP) on cAMP levels in glomeruli and tubules dissected from rat kidney. ADM (1 microM) stimulated cAMP formation in glomeruli (4-fold), cortical thick ascending limb (CTAL, 6-fold), and in the distal convoluted tubule (DCT, 16-fold). In glomeruli, ADM was more potent than CGRP [50% effective concentration (EC50) = 33.7 vs. 156 nM], whereas the opposite was true in the DCT (CGRP, EC50 = 55 vs. 327 nM for ADM). In both glomeruli and DCT, the responses to maximum concentrations of ADM and CGRP were not additive. In glomeruli, the CGRP antagonist, CGRP-(8-37), had no effect on ADM-induced increases in cAMP and inhibited CGRP activity only at concentrations > 1 microM. However, in the DCT and CTAL, CGRP-(8-37) inhibited both ADM- and CGRP-induced increases in cAMP. We conclude that in the kidney, as in other tissues, ADM and CGRP stimulate cAMP accumulation. Our results suggest that ADM and CGRP may be important in the regulation of glomerular and distal tubule function and are consistent with the view that glomeruli have "ADM-like" receptors that have a high affinity for ADM but a low affinity for CGRP and CGRP-(8-37), whereas the DCT has "CGRP-like" receptors with high affinity for CGRP and CGRP-(8-37) but a lower affinity for ADM.
Interaction of nonpeptide angiotensin II receptor antagonists with the urate transporter in rat renal brush-border membranes.
The angiotensin II (AII) antagonist, losartan, increases uric acid excretion when administered to humans. However, the active metabolite of losartan, EXP 3174, and other nonpeptide AII antagonists such as eprosartan and SB 203220 are devoid of uricosuric activity. To investigate the mechanism of losartan-induced uricosuria, we examined the effects of losartan, EXP 3174, eprosartan and SB 203220 on OH- -dependent [14C]urate uptake into rat proximal tubule brush-border membrane vesicles. Losartan (10 microM) inhibited [14C]urate uptake at all time points examined, except at equilibrium (2 hr). Losartan had no effect on urate uptake in the absence of an OH- gradient. The inhibitory effect of losartan on urate uptake was concentration dependent (IC50 = 9.5 +/- 1.4 microM) and competitive in nature. The other AII antagonists also inhibited urate uptake but were 6-8-fold less potent than losartan with IC50 values of EXP 3174 (65 +/- 13 microM), eprosartan (60 +/- 7.0 microM) and SB 203220 (74 +/- 12.5 microM). In contrast to the effects of the nonpeptide AII antagonists, the peptide antagonist, Sar1,Ile8-AII, as well as AII itself had no effect on urate uptake. These results suggest that the uricosuric activity of losartan is, at least in part, due to inhibition of urate reabsorption in the proximal tubule and is unrelated to AII receptor activity. Furthermore, losartan has a greater affinity for the urate/anion exchanger than the other AII antagonists tested. These results are in direct agreement with observations made after administration of these compounds to humans.
BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties.
The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.
Pharmacology of a potent long-acting imidazole-5-acrylic acid angiotensin AT1 receptor antagonist.
The angiotensin II antagonistic activity of SB 203220, [E-alpha-[[2-butyl-1-(4-carboxy-1-naphthalenyl)methyl]-1H- imidazol-5-yl]-methylene]-2-thiophene-propanic acid], was examined in several in vitro and in vivo assays. SB 203220 displaced [125I]angiotensin II binding from a variety of tissues including the cloned human AT1 receptor (IC(50)5-15 nM). SB 203220 (10 microM) did not interact with AT2, endothelin (ETA and ETB) or calcitonin gene-related peptide receptors. [3H]SB 203220 bound with high affinity to the AT1 receptor (Kd = 4.9 nM), but dissociated from the receptor at a much slower rate when compared to [3H]SK&F 108566. SB 203220 antagonized intracellular Ca2+ mobilization induced by angiotensin II in rat vascular smooth muscle cells and exhibited a selective and partially insurmountable antagonism of angiotensin II-induced contraction in isolated rabbit aorta. In the aorta, SB 203220 produced a concentration-dependent parallel shift in the concentration-response curve to angiotensin II [EC30 = 5.94 +/- 1.6 10(-11) M] and depressed the maximal contractile response to angiotensin II by approximately 35%. The antagonistic effect of SB 203220 in rabbit aorta was slowly reversible compared to SK&F 108566. SB 203220 displayed no agonist activity and had no effect on the contractile responses to KCl, endothelin-1 or norepinephrine. In rats, SB 203220 at 10 mg/kg i.v. inhibited angiotensin II-induced aldosterone release. Intraduodenal or oral administration of SB 203220 (1-10 mg/kg) to conscious rats and dogs inhibited the pressor responses to exogenous angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)
Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein-1 alpha.
BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G-CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100-fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB-10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.
Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution.
The cell-surface glycoprotein vascular cell adhesion molecule-1 (VCAM-1; ref. 1) mediates intercellular adhesion by specific binding to the integrin very-late antigen-4 (VLA-4, alpha 4 beta 1; ref. 3). VCAM-1, with the intercellular adhesion molecules ICAM-1, ICAM-2, ICAM-3 and the mucosal vascular addressin MAd-CAM-1, forms an integrin-binding subgroup of the immunoglobulin superfamily. In addition to their clinical relevance in inflammation, these molecules act as cellular receptors for viral and parasitic agents. The predominant form of VCAM-1 in vivo has an amino-terminal extracellular region comprising seven immunoglobulin-like domains. Functional studies have identified a conserved integrin-binding motif in domains 1 and 4, variants of which are present in the N-terminal domain of all members of the immunoglobulin superfamily subgroup. We report here the crystal structure of a VLA-4-binding fragment composed of the first two domains of VCAM-1. The integrin-binding motif (Q38IDSPL) is highly exposed and forms the N-terminal region of the loop between beta-strands C and D of domain 1. This motif exhibits a distinctive conformation which we predict will be common to all the integrin-binding IgSF molecules. These, and additional data, map VLA-4 binding to the face of the CFG beta-sheet, the surface previously identified as the site for intercellular adhesive interactions between members of the immunoglobulin superfamily.
In vivo pharmacological characterization of the non-peptide endothelin receptor antagonist SB 209670.
1. The aim of the present study was to assess the ability of SB 209670, a high affinity non-peptide endothelin receptor antagonist (0.4 and 18 nM Kis at human cloned ETA and ETB receptors, respectively), to inhibit the haemodynamic actions of endothelin-1 in vivo. 2. Systemic administration of (+/-)-SB 209670, given either as a bolus i.v. injection or as a continuous i.v. infusion, did not alter basal haemodynamic parameters in the anaesthetized rat. 3. Infusion of (+/-)-SB 209670 (10 micrograms kg-1 min-1) selectively inhibited the depressor and carotid vasodilator response to exogenous endothelin-1: 100 micrograms kg-1 min-1 was required to inhibit significantly the biphasic haemodynamic actions of endothelin-1. The haemodynamic actions of angiotensin II and calcitonin gene-related peptide were unaltered by 100 micrograms kg-1 min-1 (+/-)-SB 209670. 4. Bolus i.v. administration of (+/-)-SB 209670 (1 mg kg-1) selectively inhibited the depressor and carotid vasodilator actions of endothelin-1: 10 mg kg-1 (+/-)-SB 209670 was required to inhibit the secondary vasoconstrictor actions of endothelin-1. 5. (+/-)-SB 209670 (10 mg kg-1) was also effective at antagonizing the pressor actions of endothelin-1 in the conscious rat for up to 3 h after intraduodenal administration thereby demonstrating that the antagonist was bioavailable upon enteric administration. This dose of (+/-)-SB 209670 did not alter basal haemodynamic parameters in the conscious rat. 6. Thus, ( +/- )-SB 209670 is an effective endothelin receptor antagonist in vivo. Using the doses defined in this study, SB 209670 may, therefore, serve as a useful tool for understanding the role of endogenous endothelin-I in the control of cardiovascular function under both physiological and pathophysiological conditions.
Characterization of 125I-endothelin-1 binding to rat and rabbit renal microvasculature.
We characterized 125I-ET-1 binding to renal microvascular membranes isolated from the rat, a species showing ETB receptor-mediated renal vasoconstriction, and the rabbit in which ET-induced renal vasoconstriction is mediated by the ETA receptor. In both species, 125I-ET-1 bound in a manner consistent with a single high-affinity site. Scatchard analysis yielded Kd and Bmax values of 20.1 +/- 0.4 pM and 1343 +/- 64 fmol/mg for the rat and 21.5 +/- 0.9 pM and 810 +/- 64 fmol/mg for the rabbit. Competition binding studies with several selective (sarafotoxin 6c, BQ123) and mixed ETA/ETB (SB 209670) ET receptor ligands showed that the renal microvasculature from both species contain ETA and ETB receptors in a proportion of 40:60. In the rat, the proportion of ETA receptors was higher in the microvasculature than in glomeruli and inner medullary collecting duct cells both of which contained > 80% ETB receptors. In the rabbit, the proportion of ETA/ETB receptors was similar in the microvasculature and inner medullary collecting duct cells (approximately 40:60), whereas glomeruli contained 80% ETB receptors. Although ET-induced renal vasoconstriction in the rat and rabbit is mediated by different ET receptor subtypes, the proportion of ETA to ETB receptors is the same in the renal microvasculature from these species.
SB 203220: a novel angiotensin II receptor antagonist and renoprotective agent.
SB 203220, [(E)-alpha-[[2-butyl-1-[(4-carboxy-1-naphthalenyl)-methyl]-1H- imidazol-5-yl]-methylene]-2-thiophene-propanic acid], is a novel nonpeptide angiotensin II receptor antagonist with significant oral activity. In the present study, we compared the cardiovascular and renal effects of SB 203220 and captopril in rats with chronic renal failure induced by 5/6 nephrectomy. Preliminary studies indicated that SB 203220 (600 ppm in the diet) and captopril (250 mg/l in drinking water) significantly attenuated the pressor activity of exogenous angiotensin II and angiotensin I, respectively. After 5/6 nephrectomy, significant hypertension was observed such that at 6 weeks, systolic blood pressure had reached 176 +/- 9 mm Hg. Both SB 203220 (128 +/- 18 mm Hg) and captopril (131 +/- 7 mm Hg) significantly attenuated the hypertension. Urinary protein excretion increased progressively after renal ablation (from 7 to 124 mg/day), and this was attenuated by both SB 203220 (32 +/- 7 mg/day) and captopril (42 +/- 6 mg/day). Assessment of serum creatinine and urea nitrogen indicated that SB 203220 but not captopril resulted in maintenance of renal function, close to that observed in control rats. Both SB 203220 and captopril attenuated the renal and left ventricular hypertrophy associated with 5/6 nephrectomy.(ABSTRACT TRUNCATED AT 250 WORDS)
SB 209670, a rationally designed potent nonpeptide endothelin receptor antagonist.
An extremely potent and highly specific non-peptide, subnanomolar endothelin (ET) receptor antagonist, SB 209670, has been synthesized and characterized. SB 209670, which was rationally designed using conformational models of ET-1, selectively inhibits binding of 125I-labeled ET-1 to cloned human ET receptor subtypes ETA and ETB (Ki = 0.2 and 18 nM, respectively). SB 209670 produces concentration-dependent inhibition of ET-1-mediated vasoconstriction in isolated vascular tissues and in vivo following either intravenous or intraduodenal administration. SB 209670 produces a dose-dependent reduction in blood pressure in hypertensive rats, protects from ischemia-induced neuronal degeneration in a gerbil stroke model, and attenuates neointima formation following rat carotid artery balloon angioplasty. SB 209670 will be useful in characterizing and classifying the physiological and pathophysiological effects of ET.
Plasminogen mutants activated by thrombin. Potential thrombus-selective thrombolytic agents.
Plasmin, the enzyme responsible for degradation of fibrin in blood clots and thus thrombolysis, is normally formed when its zymogen plasminogen is activated by cleavage of the Arg561-Val562 bond by specific plasminogen activators. We have altered the activation characteristics of plasminogen by substituting the P3, P2, and P1' cleavage site residues with sequences from thrombin-cleavable proteins to produce a novel thrombolytic agent which instead is activated by the blood clotting system. Plasminogen variants with thrombin cleavage sites from fibrinogen, the thrombin receptor, factor XIII, and factor XI were cleaved by thrombin with times to 50% cleavage of 28 h, 2.5 h, 5.7 min, and 3 min, respectively. In vitro clot lysis studies have shown that a variant in which the P3-P1' residues of plasminogen were substituted by the P7-P1' residues (Thr363-Ile370) from factor XI (T51) was sufficiently rapidly cleaved by thrombin to be activated by the endogenous thrombin produced by the coagulation cascade, resulting in rapid clot dissolution. Thrombin-activatable plasminogen therefore has the capacity to short circuit the physiological hemostatic mechanisms and produce fibrinolytic activity localized to the site of thrombin formation, that is, at the thrombus itself. The novel activation mechanism combined with the natural long circulating half-life of plasminogen gives this type of thrombolytic agent the potential for thrombus-selective plasmin generation and an extended duration of action.
Human AT1 receptor is a single copy gene: characterization in a stable cell line.
To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430 +/- 15 fmol/mg] exhibiting high affinity [KD = 0.15 +/- 0.02 nM] for [125I][SAR1, Ile8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed receptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5'-(gamma-thio) triphosphate [GTP gamma S]. Angiotensin II evoked a rapid efflux of 45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.
Interactive developmental toxicity of radiofrequency radiation and 2-methoxyethanol in rats.
Concurrent exposures to chemical and physical agents occur in the workplace; exposed workers include those involved with the microelectronics industry, plastic sealers, and electrosurgical units. Previous animal research indicates that hyperthermia induced by an elevation in ambient temperature can potentiate the toxicity and teratogenicity of some chemical agents. We previously demonstrated that combined exposure to radiofrequency (RF; 10 MHz) radiation, which also induces hyperthermia and is teratogenic to exposed animals, and the industrial solvent, 2-methoxyethanol (2ME), produces enhanced teratogenicity in rats. The present study replicates and extends the previous research investigating the enhanced teratogenicity of combined RF radiation and 2ME exposures. The interactive dose-related teratogenicity of RF radiation (sham exposure or maintaining colonic temperatures at 42.0 degrees C for 0, 10, 20, or 30 min) and 2ME (0, 75, 100, 125, or 150 mg/kg) was investigated by administering various combinations of RF radiation and 2ME to groups of rats on gestation days 9 or 13; gestation-day 20 fetuses were examined for external, skeletal, and visceral malformations. The results are consistent with and extend our previous research findings. Synergism was observed between RF radiation and 2ME for some treatment combinations, but not for others. The study also clarified which gestational periods, RF radiation exposure durations, and 2ME doses would be most informative in future interaction studies to determine the lowest interactive effect level. Day 9 exposures generally evidenced little effect by 2ME, either by itself or in combination with RF radiation. In contrast, day 13 exposures resulted in highly significant effects from 2ME and RF radiation. The structures showing strong evidence of effects from both 2ME and RF radiation after exposure on gestation day 13 were the forepaw digits, forepaw phalanges, hindpaw digits, hindpaw phalanges, hind limbs, metacarpals, and metatarsals. Statistical analyses did not show a global synergistic effect, but did show evidence for a synergistic effect at intermediate levels of the dose ranges. Future research will address potential interactions at lower doses.