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R T Miller

Publications and source records attributed to R T Miller.

At least 19 recordsLinked to original sources
PubMed2006

HIV infection changes glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties.

In addition to forming the selective filtration barrier for the renal glomerulus, podocytes maintain glomerular capillary architecture by opposing distending hemodynamic forces. To understand the relationship of cytoskeletal properties and the mechanical characteristics of podocytes, we studied filamin expression and distribution and measured cell membrane deformability in conditionally immortalized wild-type (WT) mouse podocytes, and in podocytes derived from a mouse model of HIV-associated nephropathy (HIVAN). In the WT cells, filamin and F-actin were localized at the periphery and in prominent stress fibers. In the HIVAN cells, filamin expression was reduced, and stress fibers were sparse. In a microaspiration assay, HIVAN cells ruptured under minimal negative pressure. Atomic force microscopy demonstrated that the WT cells had a stiffness of 17 kPa, whereas the value for the HIVAN cells was 4 kPa. These results demonstrate that the mechanical properties of WT and HIVAN podocytes are markedly different in a manner that is consistent with differences in the composition and arrangement of their cytoskeletons. The mechanical properties of the WT podocytes suggest that these cells can better maintain capillary integrity than the HIVAN podocytes and implicate pathological assembly of the cytoskeleton as a mechanism of HIVAN.

Actinin↗
PubMed2006

Dysregulation of renal sodium transporters in gentamicin-treated rats.

We aimed to investigate the molecular mechanisms underlying the renal wasting of Na(+), K(+), Ca(2+), and Mg(2+) in gentamicin (GM)-treated rats. Male Wistar rats were injected with GM (40 or 80 mg/kg/day for 7 days, respectively; GM-40 or GM-80). The expression of NHE3, Na-K-ATPase, NKCC2, ROMK, NCC, alpha-, beta- and gamma-ENaC, and CaSR was examined in the kidney by immunoblotting and immunohistochemistry. Urinary fractional excretion of Na(+), K(+), Ca(2+), and Mg(2+) was increased and urinary concentration was decreased in both GM-40 and GM-80 rats. In cortex and outer stripe of outer medulla (cortex) in GM-80 rats, the expression of NHE3, Na-K-ATPase, and NKCC2 was decreased; NCC expression was unchanged; and CaSR was upregulated compared to controls. In the inner stripe of outer medulla (ISOM) in GM-80 rats, NKCC2 and Na-K-ATPase expression was decreased, whereas CaSR was upregulated, and NHE3 and ROMK expression remained unchanged. In GM-40 rats, NKCC2 expression was decreased in the cortex and ISOM, whereas NHE3, Na-K-ATPase, CaSR, ROMK, and NCC abundance was unchanged in both cortex and ISOM. Immunoperoxidase labeling confirmed decreased expression of NKCC2 in the thick ascending limb (TAL) in both GM-80- and GM-40-treated rats. Immunoblotting and immunohistochemical analysis revealed increased expression of alpha-, beta-, and gamma-ENaC in cortex in GM-80 rats, but not in GM-40 rats. These findings suggest that the decrease in NKCC2 in TAL seen in response to low-dose (40 mg/kg/day) gentamicin treatment may play an essential role for the increased urinary excretion of Mg(2+) and Ca(2+), and play a significant role for the development of the urinary concentrating defect, and increased urinary excretion of Na(+) and K(+). At high-dose gentamicin, both proximal and TAL sodium transporter downregulation is likely to contribute to this.

Animals↗
PubMed2003

The Hemophilia Utilization Group Study (HUGS): determinants of costs of care in persons with haemophilia A.

OBJECTIVE: The main objective of this study was to examine factors associated with utilization and costs for persons with haemophilia. STUDY DESIGN: Utilization data and patient characteristics were collected through medical record review of 336 patients receiving treatment for at least 90% of their haemophilia care at one of five comprehensive haemophilia treatment centres in California. PRINCIPAL FINDINGS: The range of factor VIII deficiency in our sample was similar to the distribution among haemophilic patients in the Western United States; 215 (64%) had severe FVIII deficiency. The mean age in our sample was 21.4 (SD = 16.2) years old and 114 (34%) were HIV-positive. In the multivariate model predicting the total cost of health care during 1995 (adjusted R2 = 0.40), total annual costs were significantly (P < 0.05) associated with being HIV-seropositive, infusing FVIII concentrate through a port vs. i.v. infusion, the number of comorbidities, moderate arthropathy (compared with no arthropathy), mild arthropathy, history of inhibitor to FVIII, and current prophylactic FVIII concentrate infusion. CONCLUSION: As expected, total health-care costs were correlated with comorbid medical conditions, such as HIV and sequelae of haemophilia such as arthropathy. Health policy should consider risk adjustment for the presence of complications such as arthropathy and HIV infection in the financing of haemophilia treatment to promote more equitable delivery of these services.

Adolescent↗
PubMed2002

Haemophilia Utilization Group Study: assessment of functional health status in haemophilia.

The purpose of this study was to assess the relationship between health care and utilization of that health care, and to provide a base measurement of health status in patients with haemophilia. Provider interview and retrospective chart review of 336 patients with haemophilia treated during 1995 at one of five comprehensive haemophilia treatment centres was conducted to measure patient health status characteristics and utilization of health care. Two health status scales were included. The first, the Self-Care Measure, was a four-point single item scale measuring the patient's ability for basic self-care, which was scored by a chart review and an interview with the health-care provider. The second, the Haemophilia Utilization Group Study (HUGS) Functional Status Measure, is a four-item, 10-point scale developed specifically for patients with haemophilia. Our sample represents 27% of actively treated patients in region IX. The mean score on the HUGS Functional Status Measure was 8.7 (SD=2.4). The HUGS scale exhibited a ceiling effect across all four scales: attitude (n=269, 80.1%), overall wellbeing (n=263, 78.3%), working (n=254, 75.6%) and orthopaedic status (n=195, 58.0%). Both higher total health-care costs and factor VIII annual costs were significantly associated with lower scores on the HUGS Functional Status Measure. Health status is a critical component in the assessment of the utilization and outcomes of care. In the absence of the availability of a patient interview, the HUGS Functional Status Measure can be used as one characteristic that explains the variation in the utilization of health care by patients with haemophilia.

Activities of Daily Living↗
PubMed2001

Characterization of hydride transfer to flavin adenine dinucleotide in neuronal nitric oxide synthase reductase domain: geometric relationship between the nicotinamide and isoalloxazine rings.

Based on the similarity in both structure and function of the reductase domain of neuronal nitric oxide synthase (nNOSred) to that of NADPH-cytochrome P450 reductase (CPR), we determined whether the characteristics of hydride transfer from NADPH to flavin adenine dinucleotide (FAD) were similar for both proteins. Secondly, we questioned whether hydride transfer from NADPH to either nNOSred or holo-nNOS was rate limiting for reactions catalyzed by these two proteins. Utilizing 500 MHz proton NMR and deuterated substrate, we determined that the stereospecificity of hydride transfer from NADPH and the conformation of the nicotinamide ring around the glycosidic bond were similar between CPR and nNOSred. Specifically, nNOSred abstracts the A-side hydrogen from NADPH, and the nicotinamide ring is in the anti conformation. We determined that the rate of hydride transfer to FAD appears to become partially rate limiting only for exceptionally good electron acceptors such as cytochrome c. Hydride transfer is not rate limiting for NO. production under any conditions used in this study. Interestingly, the deuterium isotope effect was decreased in the cytochrome c reductase assay with both nNOS and nNOSred when the assays were conducted in high ionic strength buffer, suggesting an increase in the rate of hydride transfer to FAD. These results are in stark contrast to results obtained with CPR (D. S. Sem and C. B. Kasper, 1995, Biochemistry 34, 3391-3398) whereby hydride transfer is partially rate limiting at high, but not at low, ionic strength. The seemingly opposite results in deuterium isotope effect observed with CPR and nNOSred, under conditions of high and low ionic strength, suggest differences in structure and/or regulation of these important flavoproteins.

Animals↗
PubMed2001

Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein.

In many cases, the biologic responses of cells to extracellular signals and the specificity of the responses cannot be explained solely on the basis of the interactions of known signaling proteins. Recently, scaffolding and adaptor proteins have been identified that organize signaling proteins in cells and that contribute to the nature and specificity of signaling pathways. In an effort to identify proteins that might organize the signaling system(s) activated by the extracellular Ca(2+) receptor (CaR), we used a bait construct representing the intracellular C terminus of the human CaR and the yeast two hybrid system to screen a human kidney cDNA library. We identified a clone representing the C-terminal 1042 amino acids (aa) of the cytoskeletal protein filamin (ABP-280). Analysis of truncation and deletion constructs of the CaR C terminus and the filamin cDNA clone demonstrated that the CaR and filamin interact via regions containing aa 907-997 of the CaR C terminus and aa 1566-1875 of filamin. Interaction of the two proteins in mammalian HEK-293 cells was demonstrated by co-immunoprecipitation and colocalization of them using immunofluorescence microscopy. The functional importance of their interaction was documented by transiently expressing the CaR in M2 melanoma cells that lack filamin, or in A7 melanoma cells that stably express filamin, and demonstrating that the CaR activated ERK only in the presence of filamin. Co-expression of the CaR with a peptide derived from the region of the CaR C terminus that interacts with filamin reduced the ability of the CaR to activate p42ERK in a dose-dependent manner, but did not inhibit the ability of the ET(A) receptor to activate ERK. The fact that filamin interacts with the CaR and other cell signaling proteins including mitogen-activated protein kinases and small GTPases, indicates that it may act as a scaffolding protein to organize cell signaling systems involving the CaR.

Amino Acid Sequence↗
PubMed2001

The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway.

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.

Arachidonic Acid↗
PubMed2001

The kinetic and spectral characterization of the E. coli-expressed mammalian CYP4A7: cytochrome b5 effects vary with substrate.

The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins. CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E. coli by adding the first 10 codons of CYP17alpha producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies. CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min(-1) and corresponding Km values of 74.5, 27, and 16.7 microM, respectively, in the presence of cytochrome b5. In the absence of cytochrome b5, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min(-1) and corresponding Km values of 3.9 and 33 microM, respectively. Arachidonate was not metabolized in the absence of cytochrome b5. Saturation kinetics studies performed with heme-depleted cytochrome b5 (apo cytochrome b5) yielded turnover numbers of 118 and 74 min(-1) and Km values of 74 and 25 microM with laurate and myristate, respectively, indicating that cytochrome b5 is not involved in electron transfer but rather plays a conformational role. Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (KS) in the absence and presence of cytochrome b5 (13 and 43 microM, respectively). In stopped-flow kinetics experiments, the flavin reduction (approximately 90 s(-1)) and heme reduction (approximately 9 s(-1)) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b5. Estimations of the rate of CPR (0.3 s(-1)) or cytochrome b5 (9.1 s(-1)) binding with CYP4A7 were also determined.

Animals↗
PubMed2001

Smooth muscle myosin heavy chain and caldesmon expression in the anterior vaginal wall of women with and without pelvic organ prolapse.

OBJECTIVE: The aim of this study was to quantify the expression of smooth muscle myosin heavy chain (SM-MHC) and caldesmon in the anterior vaginal wall of women with and without pelvic organ prolapse. STUDY DESIGN: Immunoblot analysis was conducted on protein extracts from the vaginal muscularis of 15 women with and 11 women without pelvic organ prolapse by using specific antibodies for SM-MHC, nonmuscle MHC-B, and caldesmon. The fraction of muscularis containing smooth muscle was determined by morphometric analysis of histologic cross sections. Reverse transcriptase-polymerase chain reaction was used to amplify SM-MHC isoforms produced by alternative splicing in the myosin head. RESULTS: Whereas the expression of SM-MHC was increased modestly (2-fold), expression of smooth muscle caldesmon was increased 6- to 7-fold in vaginal muscularis from women with prolapse. The relative distribution of SM-MHC isoforms was similar in both groups. CONCLUSIONS: Caldesmon is increased substantially in vaginal smooth muscle of women with pelvic organ prolapse. Caldesmon inhibits actin-activated myosin magnesium adenosine triphosphatase activity and inhibits the maintenance of contractile force. Thus, this disproportionate increase in caldesmon, relative to myosin, may result in inhibition of vaginal smooth muscle contractility and force maintenance.

Adult↗
PubMed2001

Regulation of cyclic guanosine monophosphate-dependent protein kinase in human uterine tissues during the menstrual cycle.

Contractility of uterine smooth muscle is essential for the cyclic shedding of the endometrial lining and also for expulsion of the fetus during parturition. The nitric oxide (NO)-cGMP signaling pathway is involved in smooth muscle relaxation. The downstream target of this pathway essential for decreasing cytoplasmic calcium and muscle tone is the cGMP-dependent protein kinase (PKG). The present study was undertaken to localize expression of PKG in tissues of the female reproductive tract and to test the hypothesis that uterine smooth muscle PKG levels vary with the human menstrual cycle. Immunohistochemistry was used to localize PKG in myometrium, cervix, and endometrium obtained during proliferative and secretory phases. The PKG was localized to uterine and vascular smooth muscle cells in myometrium, stromal cells in endometrium, and a small percentage of cervical stromal cells. Using Western blot analysis and protein kinase activity assays, the expression of PKG was reduced significantly in progesterone-dominated uteri compared with myometrium from postmenopausal women or women in the proliferative phase. These findings support a role for PKG in the control of uterine and vascular smooth muscle contractility during the menstrual cycle.

Adult↗
PubMed2001

Reorganization of myofilament proteins and decreased cGMP-dependent protein kinase in the human uterus during pregnancy.

Excessive or premature contractions of uterine smooth muscle may contribute to preterm labor. Contractile stimuli induce myosin and actin filament interactions through calcium-dependent myosin phosphorylation. The mechanisms that maintain myometrial quiescence until term are not well established, but may include control of calcium levels by nitric oxide and cGMP signaling and thin filament (caldesmon and calponin) regulation. Previously, we reported that myometrial tissues from pregnant rats are not responsive to cGMP due to decreases in cGMP-dependent protein kinase. Considering the well documented differences in the endocrinology of parturition among species, this study was conducted to test the hypothesis that the levels and subcellular distribution of caldesmon, calponin, and cGMP-dependent protein kinase are regulated with the hormonal milieu of human pregnancy. Whereas cGMP-dependent protein kinase was significantly reduced in the human uterus during pregnancy, caldesmon expression was significantly increased, and both caldesmon and calponin were redistributed to a readily extractable subcellular pool. These data suggest that cGMP-dependent protein kinase does not mediate gestational quiescence. Redistribution of thin filament-associated proteins, however, may alter uterine smooth muscle tone or the cytoskeletal framework of myocytes to maintain gestation despite the substantial distention that accompanies all intrauterine pregnancies.

Actin Cytoskeleton↗
PubMed2000

Identification of unique amino acids that modulate CYP4A7 activity.

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.

Amino Acid Sequence↗
PubMed2000

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin.

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20-40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.

Amino Acid Sequence↗
PubMed2000

The C terminus of mouse macrophage inducible nitric-oxide synthase attenuates electron flow through the flavin domain.

The sequences of nitric-oxide synthase (NOS) flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR), with the exception of a few regions. One such region is the C terminus; all NOS isoforms are 20-40 amino acids longer than CPR, forming a "tail" that is absent in CPR. To investigate its function, we removed the 21-amino acid C-terminal tail from murine macrophage inducible NOS (iNOS) holoenzyme and from a flavin domain construct. Both the truncated holoenzyme and reductase domain exhibited cytochrome c reductase activities that were 7-10-fold higher than the nontruncated forms. The truncated holoenzyme catalyzed NO formation approximately 20% faster than the intact form. Using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins and from the flavin to the heme domain is 2-5-fold faster in the absence of the C-terminal tail. The heme-nitrosyl complex, formed in all NOS isoforms during NO catalysis, is 5-fold less stable in truncated iNOS. Although both CPR and intact NOS can exist in a stable, one electron-reduced semiquinone form, neither the truncated holoenzyme nor the truncated flavin domain demonstrate such a form. We propose that this C-terminal tail curls back to interact with the flavin domain in such a way as to modulate the interaction between the two flavin moieties.

Animals↗
PubMed2000

Apoptosis, mitosis and cyclophilin-40 expression in regressing peroxisome proliferator-induced adenomas.

Chronic exposure to peroxisome proliferators (PP), including certain industrial and pharmaceutical chemicals, causes liver cancer in rodents. Continuous exposure to PP is needed for tumor development since the frequency of hepatocellular neoplasms is decreased in animals returned to control diet. To determine cellular and molecular events responsible for enhanced growth in PP-induced liver tumors, we evaluated the relationships of WY-14,643 levels, apoptosis, mitosis and cyclophilin-40 (Cyp-40) expression in regressing tumors induced by WY-14,643, a potent PP. Male F344 rats were fed WY-14,643 (0.1%) in the diet for 43 weeks and then switched to control diet for 2, 3, 5 or 36 days. Mean serum and hepatic concentrations of WY-14,643 were decreased as early as 2 days following removal of WY-14,643 as compared with rats continuously fed WY-14,643. Adenomas from rats maintained on WY-14,643 markedly compressed surrounding parenchyma. Evidence of adenoma regression was observed by 3 days of WY-14,643 withdrawal and was characterized by loss of compression. Decreased compression corresponded to increases in the apoptotic index and decreases in the mitotic index in regressing adenomas at 2, 3, and 5 days following the switch to control diet. Cyclophilins are multifunctional receptor proteins involved in numerous signal transduction pathways, including those mediated by cyclosporin, a liver tumor promoter in rats. Cyp-40 expression was markedly increased in adenomas from continuously exposed rats, but expression returned to levels similar to surrounding parenchyma in adenomas after 5 days of WY-14,643 withdrawal. Taken together, these results indicate that WY-14, 643-induced adenomas regress rapidly following withdrawal of the PP in association with declining liver WY-14,643 levels, suggesting that peroxisome proliferator-activated receptor alpha may mediate PP-induced alterations in mitogenic and/or apoptotic regulation in growing tumors, in conjunction with alterations in Cyp-40 signal transduction.

Adenoma↗
PubMed2000

Fixation and epitope retrieval in diagnostic immunohistochemistry: a concise review with practical considerations.

Diagnostic immunohistochemistry (DIHC) is an extremely valuable technological development that has revolutionized the practice of surgical pathology over the past 25 years. Like any laboratory procedure, however, the precise concatenation of methodological details that are used by any given investigator has a major effect on final results. In this overview, the authors consider several of these variables and illustrate how they may be optimized in an effective and efficient approach to quality assurance in the DIHC laboratory. The great majority of this discussion concerns processing factors as they affect paraffin section-based immunohistology because that is still, by far, the most common adjunctive morphologic procedure (AMP) used in hospital pathology. However, information is also presented on elements of tissue processing that affect other analyses as well. The material presented here is a direct reflection of the conjoint experiences of the authors. Although we discuss our preferences regarding many technical aspects of this discipline, we certainly do not mean to imply that ours are the only methods that should be utilized. Nevertheless, it is hoped that they may serve to crystallize basic concepts surrounding fixation, processing, and optimal performance of AMPs.

Biotin↗
PubMed2000

Extracellular Ca(2+)-sensing receptor is a promiscuous divalent cation sensor that responds to lead.

The extracellular Ca(2+)-sensing receptor (CaR) responds to polycations, including Ca(2+) and neomycin. This receptor is a physiological regulator of systemic Ca(2+) metabolism and may also mediate the toxic effects of hypercalcemia. A number of divalent cations, including Pb(2+), Co(2+), Cd(2+), and Fe(2+), are toxic to the kidney, brain, and other tissues where the CaR is expressed. To determine which divalent cations can activate the CaR, we expressed the human CaR in HEK-293 cells and measured activation of phospholipase A(2) (PLA(2)) and the mitogen-activated protein kinase p42ERK in response to potential agonists for the receptor. HEK-293 cells expressing the nonfunctional mutant CaR R796W served as controls. Extracellular Ca(2+), Ba(2+), Cd(2+), Co(2+), Fe(2+), Gd(3+), Ni(2+), Pb(2+), and neomycin activated the CaR, but Hg(2+) and Fe(3+) did not. We analyzed the kinetics of activation of p42ERK and PLA(2) by the CaR in response to Ca(2+), Co(2+), and Pb(2+). The EC(50) values ranged from approximately 0.1 mM for Pb(2+) to approximately 4.0 mM for Ca(2+). The Hill coefficients were >3, indicating multiple cooperative ligand binding sites or subunits. Submaximal concentrations of Ca(2+) and Pb(2+) were additive for activation of the CaR. The EC(50) for Ca(2+) or Pb(2+) was reduced four- to fivefold by the presence of the other ion. These divalent cations also activated PLA(2) via the CaR in Madin-Darby canine kidney cells that stably express the CaR. We conclude that many divalent cations activate the CaR and that their effects are additive. The facts that the CaR is a promiscuous polycation sensor and that the effects of these ions are additive to activate it suggest that the CaR may contribute to the toxicity of some heavy metals such as Pb(2+), Cd(2+), Co(2+), and Fe(2+) for the kidney and other tissues where it is expressed.

Animals↗