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1.
Bone Rep ; 18: 101672, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37064000

ABSTRACT

Lead (Pb) toxicity is a major health problem and bone is the major reservoir. Lead is detrimental to bone, affects bone remodeling and is associated with elderly fractures. Osteocalcin (OC) affects bone remodeling, improves fracture resistance and decreases with age and in some diseases. The effect of lead in osteocalcin depleted bone is unknown and of interest. We compared bone mineral properties of control and Pb exposed (from 2 to 6 months) femora from female adult C57BL6 OC+/+ and OC-/- mice using Fourier Transform Infrared Imaging (FTIRI), Micro-computed tomography (uCT), bone biomechanical measurements and serum turnover markers (P1NP, CTX). Lead significantly increased turnover in OC+/+ and in OC-/- bones producing increased total volume, area and marrow area/total area with decreased BV/TV compared to controls. The increased turnover decreased mineral/matrix vs. Oc+/+ and increased mineral/matrix and crystallinity vs. OC-/-. PbOC-/- had increased bone formation, cross-sectional area (Imin) and decreased collagen maturity compared OC-/- and PbOC+/+. Imbalanced turnover in PbOC-/- confirmed the role of osteocalcin as a coupler of formation and resorption. Bone strength and stiffness were reduced in OC-/- and PbOC-/- due to reduced material properties vs. OC+/+ and PbOC+/+ respectively. The PbOC-/- bones had increased area to compensate for weaker material properties but were not proportionally stronger for increased size. However, at low lead levels osteocalcin plays the major role in bone strength suggesting increased fracture risk in low Pb2+ exposed elderly could be due to reduced osteocalcin as well. Years of low lead exposure or higher blood lead levels may have an additional effect on bone strength.

2.
Bone ; 128: 115031, 2019 11.
Article in English | MEDLINE | ID: mdl-31401301

ABSTRACT

Osteocalcin is one of the most abundant noncollagenous proteins in bone. Phenotypes of osteocalcin knock-out mice (OC-/-) may vary on different backgrounds and with sex. Previous studies using adult female (OC-/-) mice on a mixed genetic background (129/B6) showed osteocalcin inhibited bone formation leading to weaker bone in wild-type (OC+/+). Yet on a pure (B6) genetic background male mice revealed osteocalcin improved fracture resistance and OC-/- bones were more prone to fracture. Osteocalcin is decreased with age and in some diseases (diabetes) where bone weakness is observed. The effect of osteocalcin in adult female bone from mice on a pure B6 background is unknown. We investigated differences in bone mineral properties and bone strength in female adult (6 months) (OC+/+) and (OC-/-) mice on a pure C57BL/6J background using Fourier Transform Infrared Imaging (FTIRI), micro-computed tomography (uCT), biomechanical measurements, histomorphometry and serum turnover markers (P1NP, CTX). Similar to female age matched mice on the (129/C57) background we found B6 OC-/- mice had a higher bone formation rate, no change in bone resorption, more immature mineral, decreased crystallinity and increased trabecular bone as compared to OC+/+. In contrast, the OC-/- mice on a pure B6 background had a lower bone mineral density, lower mineral to matrix ratio resulting in reduced stiffness and weaker bone strength. Our results demonstrate some properties of the OC-/- phenotype are dependent on genetic background. This may suggest that reduced osteocalcin may contribute to fracture and weaker bone in some groups of elderly and adults with diseases where osteocalcin is low.


Subject(s)
Calcification, Physiologic/physiology , Osteocalcin/metabolism , Animals , Bone Density/physiology , Calcification, Physiologic/genetics , Female , Mice, Inbred C57BL , Mice, Knockout , Osteocalcin/genetics , Spectroscopy, Fourier Transform Infrared , Stress, Mechanical , X-Ray Microtomography
3.
Arch Biochem Biophys ; 526(1): 1-8, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-22705201

ABSTRACT

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of functional connexin molecules contains a flexible turn around G12 (Arch. Biochem. Biophys.490:9,2009) allowing the N-terminus to form a portion of the channel pore near the cytoplasmic entrance. The mutants of nonfunctional connexin molecules G12S and G12Y were found to prevent this turn. Previous functional studies of loci at which Cx32 mutations cause a peripheral neuropathy, Charcot-Marie-Tooth disease, have shown that G12S is not plasma membrane inserted. Presently, we solve the structure of nonfunctional Connexin 32 mutants W3D and Y7D which do not appear to be membrane inserted. Using 2D (1)H NMR, we report that similar to G12S and G12Y, alterations in hydrophobic sidechain interactions disrupt (Y7D) or constrain (W3D) the flexible turn around G12. The alteration in the open turn around residue 12, observed in all nonfunctional mutants G12S, G12Y, W3D and Y7D correlates with loss of function. We propose that loss of the open turn causes the N-terminus to extend out of the channel pore and this misfolding may target mutants for destruction in the endoplasmic reticulum.


Subject(s)
Connexins/chemistry , Connexins/genetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutation , Nuclear Magnetic Resonance, Biomolecular , Amino Acid Sequence , Connexins/metabolism , Models, Molecular , Molecular Sequence Data , Mutant Proteins/metabolism , Peptide Fragments/chemistry , Protein Conformation , Solutions , Gap Junction beta-1 Protein
4.
Bone ; 47(5): 888-94, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20643234

ABSTRACT

Lead toxicity is a significant problem in the U.S. with elevated blood lead levels being highest among very young children and older adults >50 years old. Bone is the major reservoir of body lead, accounting for 75% in children and 90% in adults. Very little is known about the effect of lead on bone mineral properties in adults. We investigated the effect of lead on the femora from adult, 6 month old female C57/BL6 mice who were administered lead in the drinking water (250 ppm, blood lead 33 µg/dL) for 4 months. Bone mineral properties were examined using Fourier Transform Infrared Microscopy (FTIRM), quantitative microcomputed tomography (microCT) and whole bone mechanical testing. Lead significantly decreased the bone mineral density in the cortical and proximal cancellous bone and increased the marrow area in the cortical bone with microCT. Whole bone three-point bending showed a trend of decreased maximum and failure moments in the lead treated bones compared to controls. Lead significantly decreased the mineral/matrix ratio, collagen maturity and crystallinity in the trabecular bone as measured by FTIRM. In the cortical bone lead significantly decreased collagen maturity and bone crystal size by FTIRM. In contrast to cell culture studies, lead significantly increased serum osteocalcin levels. Lead also significantly increased the bone formation and resorption markers suggesting increased bone turnover. These data show that lead increases bone turnover resulting in weaker cortical bone in adult female mice and suggest that lead may exacerbate bone loss and osteoporosis in the elderly.


Subject(s)
Bone Density/drug effects , Lead/toxicity , Animals , Biomechanical Phenomena/physiology , Calcification, Physiologic/drug effects , Female , Mice , Mice, Inbred C57BL , Organometallic Compounds/toxicity , Osteocalcin/metabolism , Osteoporosis/chemically induced , Spectroscopy, Fourier Transform Infrared , X-Ray Microtomography
5.
Arch Biochem Biophys ; 490(1): 9-16, 2009 Oct 01.
Article in English | MEDLINE | ID: mdl-19638273

ABSTRACT

The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of a representative connexin molecule contains a flexible turn around glycine 12 [P.E. Purnick, D.C. Benjamin, V.K. Verselis, T.A. Bargiello, T.L. Dowd, Arch. Biochem. Biophys. 381 (2000) 181-190] allowing the N-terminus to reside at the cytoplasmic entry of the channel forming a voltage-sensor. Previous functional studies or neuropathies have shown that the mutation G12Y and G12S form non-functional channels while functional channels are formed from G12P. Using 2D (1)H NMR we show that similar to G12, the structure of the G12P mutant contains a more flexible turn around residue 12, whereas the G12S and G12Y mutants contain tighter, helical turns in this region. These results suggest an unconstrained turn is required around residue 12 to position the N-terminus within the pore allowing the formation of the cytoplasmic channel vestibule, which appears to be critical for proper channel function.


Subject(s)
Connexins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Amino Acid Sequence , Animals , Connexins/chemical synthesis , Connexins/genetics , Connexins/isolation & purification , Hydrophobic and Hydrophilic Interactions , Ion Channel Gating/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Conformation , Protein Structure, Secondary/genetics , Gap Junction beta-1 Protein
6.
Biochim Biophys Acta ; 1784(11): 1534-45, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18793762

ABSTRACT

Structural information on the effect of Pb(2+) on proteins under physiologically relevant conditions is largely unknown. We have previously shown that low levels of lead increased the amount of osteocalcin bound to hydroxyapatite (BBA 1535:153). This suggested that lead induced a more compact structure in the protein. We have determined the 3D structure of Pb(2+)-osteocalcin (49 amino acids), a bone protein from a target tissue, using (1)H 2D NMR techniques. Lead, at a stoichiometry of only 1:1, induced a similar fold in the protein as that induced by Ca(2+) at a stoichiometry of 3:1. The structure consisted of an unstructured N-terminus and an ordered C-terminal consisting of a hydrophobic core (residues 16-49). The genetic algorithm-molecular dynamics simulation predicted the lead ion was coordinated by the Gla 24 and Gla 21 residues. It is proposed that mineral binding occurs via uncoordinated Gla oxygen ions binding to calcium in hydroxyapatite. A comparison of Pb(2+)- and Ca(2+)-osteocalcin suggests Pb(2+), at a lower stoichiometry, may induce similar conformational changes in proteins and subsequent molecular processes normally controlled by calcium alone. This may contribute to a molecular mechanism of lead toxicity for calcium binding proteins. Lead exposure may alter the amount of mineral bound osteocalcin and contribute to abnormal bone remodeling.


Subject(s)
Lead Poisoning/metabolism , Lead/metabolism , Nuclear Magnetic Resonance, Biomolecular/methods , Osteocalcin/chemistry , Amino Acid Sequence , Animals , Binding Sites , Calcium/metabolism , Cattle , Hydrogen/chemistry , Lead/chemistry , Models, Molecular , Molecular Sequence Data , Osteocalcin/metabolism , Protein Binding
7.
Biochemistry ; 42(25): 7769-79, 2003 Jul 01.
Article in English | MEDLINE | ID: mdl-12820886

ABSTRACT

Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using (1)H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and (1)H and (13)C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16-49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16-49 include type III turns (residues 20-25) and two alpha-helical regions (residues 27-35 and 41-44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm-molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 A in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.


Subject(s)
Calcium/metabolism , Osteocalcin/chemistry , Animals , Cattle , Magnetic Resonance Spectroscopy , Osteocalcin/metabolism , Protein Binding
8.
Biochim Biophys Acta ; 1535(2): 153-63, 2001 Feb 14.
Article in English | MEDLINE | ID: mdl-11342004

ABSTRACT

Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb(2+) at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb(2+) on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb(2+) induces a similar structure in osteocalcin as Ca(2+) but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb(2+) to osteocalcin (K(d)=0.085 microM) than Ca(2+) (K(d)=1.25 mM). The hydroxyapatite binding assays show that Pb(2+) causes an increased adsorption to hydroxyapatite, similar to Ca(2+), but at 2-3 orders of magnitude lower concentration. Low Pb(2+) levels (1 microM) in addition to physiological Ca(2+) levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca(2+) alone. These results suggest a molecular mechanism of Pb(2+) toxicity where low Pb(2+) levels can inappropriately perturb Ca(2+) regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb(2+)-intoxicated animals.


Subject(s)
Lead/pharmacology , Osteocalcin/chemistry , Animals , Calcium/pharmacology , Cations, Divalent , Cattle , Circular Dichroism , Durapatite/chemistry , Osteocalcin/metabolism
9.
Arch Biochem Biophys ; 381(2): 181-90, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-11032405

ABSTRACT

Charged amino acid residues in the amino terminus of gap junction forming proteins (connexins) form part, if not all, of the transjunctional voltage sensor of gap junction channels and play a fundamental role in ion permeation. Results from studies of the voltage dependence of N-terminal mutants predict that residues 1-10 of Group I connexins lie within the channel pore and that the N-terminus forms the channel vestibule by the creation of a turn initiated by the conserved G12 residue. Here we report that intercellular channels containing mutations of G12 in Cx32 to residues that are likely to interfere with flexibility of this locus (G12S, G12Y, and G12V) do not express junctional currents, whereas a connexin containing a proline residue at G12 (Cx32G12P), which is expected to maintain a structure similar to that of the G12 locus, forms nearly wild-type channels. We have solved the structure of an N-terminal peptide of Cx26 (MDWGTLQSILGGVNK) using 1H 2D NMR. The peptide contains two structured domains connected by a flexible hinge (domain-hinge-domain motif) that would allow the placement of the amino terminus within the channel pore. Residues 1-10 adopt a helical conformation and line the channel entrance while residues 12-15 form an open turn. Overall, there is good agreement between the structural and dynamic features of the N-terminal peptide provided by NMR and the functional studies of the voltage dependence of channels formed by wild-type and N-terminal mutations.


Subject(s)
Connexins/chemistry , Amino Acid Sequence , Animals , Connexin 26 , Connexins/genetics , Connexins/metabolism , Gap Junctions/chemistry , Humans , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Oocytes/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Static Electricity , Thermodynamics , Xenopus
10.
Biochim Biophys Acta ; 1272(3): 133-9, 1995 Dec 12.
Article in English | MEDLINE | ID: mdl-8541343

ABSTRACT

Ischemia is often implicated as a cause of acute renal failure. We have investigated the effect of various concentrations of extracellular Mg2+ on the post-ischemic recovery of ATP and low intracellular Na+ in the isolated perfused rat kidney using 31P and triple-quantum filtered (TQ) 23Na-NMR spectroscopy. Following a 1 h period of stopped flow ischemia, the kidneys exposed to 0.3 mM Mg2+ throughout the experiment exhibited a significantly (p < 0.05) decreased post-ischemic fractional recovery of ATP (56 +/- 7%) as well as a significantly (p < 0.05) increased accumulation of P(i) (250 +/- 30%) as compared to kidneys exposed to 1.2 mM Mg2+ throughout (88 +/- 5% recovery of [ATP] and 158 +/- 8% accumulation of [P(i)]). Kidneys exposed to 0.3 mM Mg2+ during the pre-ischemic and ischemic periods but to 1.2 mM Mg2+ during reperfusion also showed better recovery of ATP (83 +/- 6%) and lower accumulation of P(i) (143 +/- 8%) compared to kidneys exposed to low Mg2+ (0.3 mM) throughout the experiment. Measurements of the 23Na TQ signal following ischemia-reperfusion revealed that kidneys exposed to 1.2 mM Mg2+ exhibited significantly improved maintenance of low intracellular Na+ as compared to those exposed to 0.3 mM Mg2+ ([Na+]i = 107 +/- 7% in 1.2 mM Mg2+ vs. 152 +/- 3% in 0.3 mM Mg2+). No significant difference was found in the pre-ischemic basal intracellular free Ca2+ level (as measured by 19F-NMR in combination with 5 FBAPTA) between kidneys perfused with 1.2 mM and 0.3 mM Mg2+, and comparable depletion of ATP occurred during ischemia under both experimental conditions. These data indicate that increased extracellular Mg2+ has a protective effect against post-ischemic damage, probably related to its role in resynthesis of ATP during post-ischemic reperfusion. Our results would imply a greater vulnerability of the kidney to ischemic damage in hypomagnesemic clinical conditions such as alcoholism and diabetes.


Subject(s)
Adenosine Triphosphate/metabolism , Ischemia/metabolism , Kidney/blood supply , Kidney/metabolism , Magnesium/pharmacology , Sodium/metabolism , Animals , Extracellular Space , Intracellular Fluid/metabolism , Magnesium/blood , Magnesium/metabolism , Magnetic Resonance Spectroscopy , Perfusion , Rats , Rats, Sprague-Dawley , Reperfusion
11.
Biochim Biophys Acta ; 1226(2): 131-7, 1994 May 25.
Article in English | MEDLINE | ID: mdl-8204659

ABSTRACT

Lead, an environmental toxin, is known to impair some of the functional properties of osteocalcin, a small protein (MW, 5700) active in bone mineralization and resorption. To investigate a possible mechanism of lead toxicity at the molecular level, we have studied the interaction of lead with osteocalcin using 43Ca and 1H NMR. The measured 43Ca NMR linewidth as well as longitudinal relaxation rate (1/T1) of 43CaCl2 progressively increased with increasing amounts of added osteocalcin. A titration measuring 43Ca linewidth as a function of [Ca2+]/[Osteocalcin] ratio could be fitted to a single metal binding site with a dissociation constant of 7 microM. The 43Ca 1/T1 of Ca-osteocalcin decreased in the presence of Pb2+ due to competitive displacement of Ca2+ by Pb2+. The magnitude of decrease in the effect of osteocalcin on 43Ca 1/T1 in the presence of Pb2+ was consistent with the existence of only one tight divalent cation binding site. An analysis of the NMR T1 data in osteocalcin solutions containing both Pb2+ and Ca2+ yielded a Pb-osteocalcin dissociation constant of about 2 nM. The 1H NMR spectra showed Pb-induced changes in the same aliphatic and aromatic resonances of osteocalcin that are also affected by Ca(2+)-binding, supporting interaction of Pb2+ at the Ca2+ site. However, the existence of significant differences between the Pb-osteocalcin and Ca-osteocalcin NMR spectra indicates some differences in the structures of the two complexes. Since Pb2+ inhibits the binding of osteocalcin to hydroxyapatite, the high affinity of Pb2+ for osteocalcin would indicate significant inactivation of osteocalcin even at submicromolar free lead levels. Pb(2+)-induced inactivation of osteocalcin could affect bone mineral dynamics and may be related to the observed inverse correlation between blood Pb(2+)-levels and stature and chest circumference observed in growing children.


Subject(s)
Calcium/chemistry , Lead/toxicity , Osteocalcin/chemistry , Animals , Calcium Isotopes , Cattle , Lead/chemistry , Magnetic Resonance Spectroscopy , Osteocalcin/isolation & purification , Osteocalcin/metabolism
12.
Biochim Biophys Acta ; 1226(1): 83-8, 1994 Apr 12.
Article in English | MEDLINE | ID: mdl-8155743

ABSTRACT

We previously reported a significant derangement of intracellular free calcium ion concentration in the isolated perfused kidney of adult spontaneously hypertensive rat (SHR) (J. Biol. Chem. 267, 3637-3643, 1992). In order to investigate whether an abnormality in intracellular free calcium or another ion precedes the development of elevated blood pressure in SHR, we have now compared intracellular free Ca2+, Na+ and pH, using 31P, 19F, and triple quantum-filtered (TQ) 23Na NMR, in perfused kidneys from prehypertensive young SHR and normotensive young Wistar-Kyoto (WKY) rats (5-6 weeks old) which showed no significant difference in blood pressure (B.P. = 120 +/- 5 mmHg and 115 +/- 3 mmHg, for SHR and WKY rats, respectively). Like the adult kidney, no significant differences in intracellular ATP concentration or intracellular pH were found between young prehypertensive SHR and normotensive WKY rat kidneys. The TQ 23Na NMR signal was 47% higher in the SHR kidney, but, due to biological variability and measurement errors, this difference could not be shown to be statistically significant. However, a significant (40%; P < 0.05) increase was found in O2 consumption rate, a measure of the Na+/K(+)-ATPase activity, of the young prehypertensive SHR kidney in comparison to the age-matched WKY rat kidney (7.25 +/- 0.75 for SHR vs. 5.17 +/- 0.18 mumol O2/min g for WKY rat, n = 6). Furthermore, a highly significant (92%; P < 0.02) increase in intracellular free Ca2+ concentration was observed in kidneys from young SHR that had not yet developed high blood pressure in comparison to the kidneys from young normotensive WKY rats (648 +/- 76 nM vs. 339 +/- 39 nM, n = 4), despite the fact that there was no significant difference in blood pressure. Increased intracellular free Ca2+ thus appears to be part of a primary defect, in the prehypertensive young SHR kidney, which may, by way of increased release of arachidonic acid, and subsequent increased production of vasoconstricting arachidonic acid metabolites via the cytochrome P450 pathway, induce elevated blood pressure in the adult SHR.


Subject(s)
Calcium/metabolism , Hypertension/metabolism , Kidney/metabolism , Sodium/metabolism , Adenosine Triphosphate/metabolism , Animals , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Oxygen Consumption , Rats , Rats, Inbred SHR , Rats, Inbred WKY
13.
Biochim Biophys Acta ; 1182(3): 329-32, 1993 Oct 20.
Article in English | MEDLINE | ID: mdl-8399369

ABSTRACT

Hemodynamic and 31P-NMR spectroscopic studies were performed on intact, perfused working rat hearts exposed to low (0.3 mM) extracellular Mg([Mg2+]o). Low [Mg2+]o perfusion resulted in rapid and significant falls in cardiac output, coronary flow, stroke volume, developed pressure and the rate-pressure product. Concomitant with this O2 consumption decreased and lactate production increased. Hearts perfused with 0.3 mM, instead of 1.2 mM, [Mg2+]o exhibited significant reductions in [ATP], [PCr], intracellular free Mg ([Mg2+]i), and pHi; a marked rise in intracellular Pi corresponding to a precipitous fall in the cytosolic phosphorylation potential was seen. Reintroduction of 1.2 mM [Mg2+]o failed to reestablish either normal hemodynamics, or high-energy phosphates and intracellular Pi, suggesting irreversible myocyte injury. These observations are consistent with the tenet that low [Mg2+]o can result in marked reduction in oxygen and substrate delivery to the cardiac myocytes, probably as a result of coronary vasoconstriction.


Subject(s)
Coronary Disease/etiology , Magnesium Deficiency/complications , Magnesium/analysis , Myocardium/metabolism , Phosphates/metabolism , Animals , Hemodynamics , Hydrogen-Ion Concentration , Magnesium Deficiency/metabolism , Magnetic Resonance Spectroscopy , Male , Perfusion , Rats , Rats, Wistar
14.
J Biol Chem ; 268(2): 991-6, 1993 Jan 15.
Article in English | MEDLINE | ID: mdl-8419377

ABSTRACT

Alterations in intracellular cation concentrations in the kidney during hyperglycemia may play a role in a number of complications associated with diabetes mellitus such as renal hypertrophy and hypertension. In this study, we have investigated the effect of 25 mM glucose on intracellular pH and free Mg2+, free Ca2+, and Na+ concentrations in the perfused kidneys of Sprague-Dawley rats using 31P, 19F, and 23Na triple-quantum filtered NMR. No significant alteration occurred in the intracellular free Mg2+ concentration, pH, or ATP concentration during hyperglycemia. However, a sizable (approximately 50%) increase in the intracellular Na+ concentration was inferred from 23Na triple-quantum filtered NMR after 30-45 min of perfusion with 25 mM glucose. Intracellular free Ca2+, measured to be 390 +/- 15 nM at 5 mM glucose, increased significantly (95%; p < 0.001) to a value of 765 +/- 28 nM after 30 min of perfusion with 25 mM glucose. This effect of glucose was reversible. Only small increases (< or = 20%) in free Ca2+ were found with addition of comparable concentrations of a nontransportable sugar (20 mM mannitol), indicating that glucose entry into the cell (through the Na+/glucose cotransporter) plays a role in causing the free Ca2+ increase. No effect on free Ca2+ was however, seen with 1 mM ouabain, which caused a sizable increase in intracellular Na+, indicating that Na+/Ca2+ exchange does not play a significant role in the maintenance of low intracellular free Ca2+ in the kidney and that the observed increase in free Ca2+ is probably due to a decrease in the Ca2+/Mg(2+)-ATPase activity during hyperglycemia. Increased concentrations of Na+ and free Ca2+ during hyperglycemia may be involved in the mechanism of renal hypertrophy and hypertension, frequently associated with diabetes mellitus.


Subject(s)
Calcium/metabolism , Glucose/metabolism , Hyperglycemia/metabolism , Kidney/metabolism , Magnesium/metabolism , Phosphates/metabolism , Sodium/metabolism , Animals , Fluorine , Magnetic Resonance Spectroscopy/methods , Phosphorus , Rats , Rats, Sprague-Dawley , Reference Values
15.
J Biol Chem ; 267(6): 3637-43, 1992 Feb 25.
Article in English | MEDLINE | ID: mdl-1740416

ABSTRACT

We have investigated hypertension-associated alterations in intracellular cations in the kidney by measuring intracellular pH, free Mg2+, free Ca2+, and Na+ concentrations in perfused normotensive and hypertensive rat (8-14 weeks old) kidneys using 31P, 19F, and double quantum-filtered (DQ) 23Na NMR. The effects of both anoxia and ischemia on the 23Na DQ signal confirmed its ability to detect changes in intracellular Na+. However, there was a sizable contribution of the extracellular Na+ to the 23Na DQ signal of the kidney. The intracellular free Ca2+ concentration, measured using 19F NMR and 5,5'difluoro-1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid, also increased dramatically during ischemia; the increase could be partly reversed by reperfusion. No significant differences were found between normotensive and hypertensive kidneys in the ATP level, intracellular pH, intracellular free Mg2+, and the 23Na DQ signal or in the extent of the extracellular contribution to the 23Na DQ signal. Oxygen consumption rates were also similar for the normotensive (5.02 +/- 0.46 mumol of O2/min/g) and hypertensive (5.47 +/- 0.42 mumol O2/min/g) rat kidneys. The absence of a significant difference in intracellular pH, Na+ concentration, and oxygen consumption between normotensive and hypertensive rat kidneys suggests that an alteration in the luminal Na+/H+ antiport activity in hypertension is unlikely. However, a highly significant increase (64%, p less than 0.01) in free Ca2+ concentration was found in perfused kidneys from hypertensive rats (557 +/- 48 nM, blood pressure = 199 +/- 5 mmHg, n = 6) compared with normotensive rats (339 +/- 21 nM, blood pressure = 134 +/- 6, n = 4) indicating altered renal calcium homeostasis in essential hypertension. An increase in intracellular free Ca2+ concentration without an accompanying change in the intracellular Na+ suggests, among many possibilities, that the Ca2+/Mg(2+)-ATPase may be inhibited in the hypertensive renal tissue.


Subject(s)
Calcium/metabolism , Hypertension/metabolism , Kidney/metabolism , Magnesium/metabolism , Sodium/metabolism , Animals , Cations , Chelating Agents , Egtazic Acid/analogs & derivatives , Egtazic Acid/chemistry , Hydrogen-Ion Concentration , Ischemia/metabolism , Kidney/blood supply , Magnetic Resonance Spectroscopy , Perfusion , Rats , Rats, Inbred SHR , Rats, Inbred WKY
16.
Am J Physiol ; 261(1 Pt 2): F153-62, 1991 Jul.
Article in English | MEDLINE | ID: mdl-1858897

ABSTRACT

To test the hypothesis that growth and dietary Pi affect the intracellular concentration of Pi ([Pi]i) as well as its renal reabsorption, we measured nuclear magnetic resonance (NMR)-visible [Pi]i in isolated perfused kidneys of less than 1- and greater than 4-wk-old guinea pigs fed various amounts of Pi. Changes in [Pi]i were correlated with those in fractional Pi reabsorption (FRPi) in vivo and in capacity (Vmax) for Na(+)-Pi cotransport in microvilli derived from animals of similar age and fed the same diets. In animals fed normal (0.76% Pi) diet, [Pi]i was lower (0.91 +/- 0.14 vs. 1.85 +/- 0.23 mM, P less than 0.05), whereas FRPi was higher (0.90 +/- 0.02 vs. 0.70 +/- 0.03, P less than 0.01) in less than 1- than in greater than 4-wk-old guinea pigs. Pi deprivation decreased [Pi]i in mature animals to 0.74 +/- 0.29 mM, P less than 0.05, and increased FRPi to 0.99 +/- 0.01. Excess dietary Pi increased [Pi]i in immature animals to 1.67 +/- 0.56 mM, P less than 0.05, and decreased FRPi to 0.55 +/- 0.03. Diet-induced changes in [Pi]i were associated with reciprocal changes in Vmax of similar absolute magnitude in immature and mature animals. However, diets that resulted in comparable [Pi]i at the two ages were associated with higher (P less than 0.05) Vmax in less than 1- than in greater than 4-wk-old animals. The reciprocal nature of the relationship between [Pi]i and renal Pi transport indicates that [Pi]i is primarily determined by Pi efflux from the cells or Pi organification rather than Pi influx through Na(+)-Pi cotransport. Findings indicate that changes in [Pi]i with growth or diet may be a cause but cannot be the consequence of changes in abundance or maximal mobility of Na(+)-Pi cotransporters. Data also indicate that factors in addition to low [Pi]i contribute to the high Na(+)-Pi cotransport capacity observed in renal microvilli of growing animals.


Subject(s)
Aging/metabolism , Diet , Kidney/metabolism , Magnetic Resonance Spectroscopy , Phosphates/metabolism , Animals , Biological Transport , Guinea Pigs , In Vitro Techniques , Intracellular Membranes/metabolism , Kidney/cytology , Male , Osmolar Concentration , Perfusion , Phosphates/administration & dosage , Water/metabolism
17.
Biochim Biophys Acta ; 1092(3): 341-6, 1991 May 17.
Article in English | MEDLINE | ID: mdl-1904777

ABSTRACT

Lead has been shown to affect calcium homeostasis. However, there is no prior evidence to indicate an effect of low concentrations of lead in the environment (approximately 1 microM) on the intracellular free Ca2+ concentration in any human tissue. We have investigated the effect of lead on the intracellular free Ca2+ concentration of human blood platelets using 19F-NMR and a fluorinated intracellular Ca2+ indicator. We report a basal intracellular free Ca2+ value of 172 +/- 8 nM. Treatment with 1, 5, 10 and 25 microM Pb2+ resulted in average increases in intracellular free Ca2+ of 39%, 91%, 135% and 172%, respectively. The percent increase in intracellular free Ca2+ was linearly and positively correlated with the log of Pb2+ concentration. Using atomic absorption spectroscopy, a significant increase in total calcium of approx. 10 nmol/mg protein was found in 25 microM Pb2+ treated platelets. This indicates that influx of external Ca2+ contributes to the observed increase in free Ca2+. The results provide an explanation for the previously reported effects of lead on platelet function, and suggest a mechanism for low level lead-induced hypertension.


Subject(s)
Blood Platelets/drug effects , Calcium/blood , Lead/pharmacology , Adenosine Triphosphate/blood , Blood Platelets/metabolism , Egtazic Acid/metabolism , Humans , Hypertension/chemically induced , Intracellular Fluid/drug effects , Lead/blood , Lead/toxicity , Magnetic Resonance Spectroscopy , Spectrophotometry, Atomic
18.
Magnes Trace Elem ; 10(2-4): 99-116, 1991.
Article in English | MEDLINE | ID: mdl-1844566

ABSTRACT

The cellular bioenergetic responses of isolated perfused working rat hearts to alterations in hemodynamic function caused by acute exposure to elevated levels of extracellular magnesium ions ([Mg2+]o) were examined using 31P nuclear magnetic resonance (31P NMR) spectroscopy. Results showed that in hearts working against 90 cm H2O afterload, an increase in [Mg2+]o from 1.2 to 4.8 mM reduced the heart rate by 35%, while coronary flow was increased by 38%. Unexpectedly, despite the pronounced bradycardia, the rate-pressure product was reduced only slightly (from 2.36 x 10(4) to 2.08 x 10(4) mm Hg/min) due to a significant increase (36%) in systolic pressure. In addition, cardiac output actually increased by 23%, owing to a > 100% increase in stroke volume, indicating that the performance of the heart was improved and suggesting that the efficiency of the heart was improved as well. In a separate series of experiments, 31P NMR measurements performed on hearts perfused in the Langendorff mode revealed that elevated levels of [Mg2+]o increase phosphocreatine (PCr) levels by 23% (from 9.2 to 11.3 mM), while Pi levels declined by a corresponding amount. Perfusion of hearts in the working mode with elevated [Mg2+]o was also observed to increase PCr levels from 6.3 to 9.0 mM, while ATP levels declined by 17%. Measurement of the chemical shift difference between Pi and PCr and that between the alpha and beta phosphate resonances of ATP were used to determine intracellular pH and the cytosolic levels of free Mg2+ ([Mg2+]i), respectively. These results showed that acute exposure of hearts, perfused in either the working or Langendorff mode, to increased levels of [Mg2+]o increased intracellular pH by 0.12-0.13 units, while free Mg2+ nearly doubled to a level of 1.1-1.2 mM. The latter observation may suggest that acute variations in the level of [Mg2+]o can influence a multitude of cellular processes requiring Mg2+ as an essential cofactor. Using the above data and assuming equilibrium of the creatine kinase reaction, the levels of ADP, cytosolic phosphorylation potential ([ATP]/[ADP][Pi]) and free energy change from ATP hydrolysis (-delta G/delta E) were also calculated. Results obtained illustrate that in the presence of elevated [Mg2+]o, ADP levels declined by 33-48%, the cytosolic phosphorylation potential increased from 41 to 112 mM-1 and -delta G/delta E increased from 56.7 to 59.3 kJ/mol. These changes are not completely accountable by the known bradycardia and vasodilatory effects of elevated [Mg2+]o and strongly argue for a direct action of [Mg2+]o on the myocyte as well.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Hemodynamics/physiology , Magnesium/physiology , Myocardium/metabolism , Adenosine Triphosphate/metabolism , Animals , Energy Metabolism , Heart/physiology , Hydrogen-Ion Concentration , In Vitro Techniques , Magnesium/metabolism , Magnetic Resonance Spectroscopy , Male , Models, Biological , Myocardium/cytology , Phosphorus , Phosphorylation , Rats , Rats, Wistar
19.
J Biol Chem ; 265(34): 20833-8, 1990 Dec 05.
Article in English | MEDLINE | ID: mdl-2249990

ABSTRACT

The mechanism of lead toxicity at the cellular level remains unknown, although an effect of lead on intracellular Ca2+ has been described. Since bone is a major target for lead, we have investigated the effect of lead on bioenergetic rates and on the intracellular free Mg2+ concentration in cultured osteoblastic bone cells. Using 31P NMR and the saturation transfer technique we have detected a sizable (18%) transfer of saturation from gamma ATP to Pi in a perfused osteoblastic osteosarcoma bone cell line, Ros 17/2.8, and have found a large (greater than 82%) reduction in the Pi----ATP rate upon treatment with 10 microM Pb2+. The NMR-measured unidirectional rate was much greater than the net rate of ATP synthesis through glycolysis and oxidative phosphorylation. By using iodoacetate we investigated the mechanism of the saturation transfer and found that it is catalyzed by the glycolytic enzyme couple glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. The net rate of glycolysis as measured by lactate production and that of oxidative phosphorylation as measured by O2 consumption were found to be significantly decreased by 18 and 74%, respectively, with lead treatment. In addition, from the chemical shifts of intracellular ATP resonances, we found a significant reduction of 21% in the intracellular free Mg2+ concentration upon Pb2+ treatment. The observed lead-induced reduction in ATP synthesis/utilization and the decrease in intracellular free Mg2+ may contribute to the impairment of bone formation during lead intoxication.


Subject(s)
Lead/pharmacology , Osteoblasts/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Line , Glycolysis/drug effects , Kinetics , Magnetic Resonance Spectroscopy/methods , Osteoblasts/drug effects , Osteosarcoma , Oxygen Consumption/drug effects , Phosphates/metabolism , Phosphorus
20.
Biochim Biophys Acta ; 1054(2): 250-5, 1990 Sep 01.
Article in English | MEDLINE | ID: mdl-2169314

ABSTRACT

Using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, we have recently demonstrated that Pb2+ treatment elevates the intracellular free calcium ion concentration ([Ca2+]i) of rat osteoblastic osteosarcoma cells (ROS 17/2.8) (Proc. Natl. Acad. Sci. USA (1989) 86, 5133-5135). In this study, we have examined the effects of Pb2+ on the basal and parathyroid hormone (PTH)-stimulated levels of [Ca2+]i and cAMP in cultured ROS 17/2.8 cells. PTH treatment (400 ng/ml) stimulated a 150% elevation in [Ca2+]i from a control level of 105 +/- 25 nM to a concentration of 260 +/- 24 nM. Treatment of ROS 17/2.8 cells with Pb2+ (5 microM) alone produced a 50% elevation in the [Ca2+]i to 155 +/- 23 nM. Pb2+ treatment diminished subsequent elevation in [Ca2+]i in response to PTH administration thereby limiting the peak increase in [Ca2+]i to only 25% or 193 +/- 22 nM. In contrast to the dampening effect of Pb2+ on the peak rise in [Ca2+]i produced by PTH, Pb2+ (1 to 25 microM) had no effect on PTH-induced increments in intracellular cAMP levels. Hence, Pb2+ dissociated the PTH stimulation of adenylate cyclase from PTH effects on [Ca2+]i and shifted the regulation of [Ca2+]i beyond the control of PTH modulation. These observations further extend the hypothesis that an early toxic effect of Pb2+ at the cellular level is perturbation of [Ca2+]i homeostasis.


Subject(s)
Calcium/metabolism , Cyclic AMP/metabolism , Lead/pharmacology , Osteosarcoma/metabolism , Parathyroid Hormone/pharmacology , Second Messenger Systems/drug effects , Animals , Fluorine , Homeostasis , Magnetic Resonance Spectroscopy , Rats , Tumor Cells, Cultured
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