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Department of Oral Biology, Medical College of Georgia, School of Dentistry, Augusta, Georgia 30912-1129
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • Ca++transport • fluoride • Ca++-pump • endoplasmic reticulum Ca++-pump • plasma membrane Ca++-pump • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). High exposure levels, however,
are of concern. Skeletal fluorosis has been reported in populations
exposed to 8–10 mg F-/L for 10 y or more
(Whitford 1992
). Two epidemiological studies indicate
that the incidence of bone fractures also correlates significantly with
water fluoridation (Jacobsen et al. 1990
, Sowers et al. 1991
). The second of these two studies, by Sowers et
al., showed a significant increase in fracture incidence for women
55–80 y of age. This increase occurred independent of the confounding
variables of Ca++ intake, weight, size, age, and
hormone usage.
High F- exposures of 10 or more mg/L are
attainable from environments rich in F- from
natural or industrial sources. High F- exposure
also occurs during therapeutic exposure to F-
for the treatment of osteoporosis (Pak et al. 1997
). Of
additional concern is the potential for high exposures, particularly of
children, from the consumption of pleasant-tasting,
F--containing dental care products
(Whitford 1989).
Das and Susheela (1993)
studied the effects of long-term
administration of F- on intestinal absorption,
urinary excretion, and plasma levels of Ca++ in
rabbits and found clear systemic effects. Hypocalcemia was observed in
F- treated animals despite a drop in
Ca++ excretion and an increase in intestinal
Ca++ absorption. These findings suggest an
increase in Ca++ retention by the tissues in
chronic F- toxicity. This increase in
Ca++ retention would be consistent with the
inhibition of a cellular Ca++ efflux mechanism as
well. Andersen et al. (1986)
produced a normocalcemic condition in
F- treated pigs fed a Ca++ and
vitamin D-supplemented diet. This normocalcemic state, however, was
also associated with a retention of Ca++ by
tissues that was much higher than normal. In an effort to restore
normocalcemia in osteofluorotic subjects by increasing
Ca++, the result may actually be the induction of
osteosclerosis. Thus, osteomalacia and osteosclerosis may both be
observed in osteofluorotic subjects as a function of dietary
Ca++.
Long-term exposure to elevated levels of F- has
negative effects on calcium-dependent processes in the kidney as
well. For example, studies have shown varying degrees of tubular
dysfunction associated with high F- exposure
(Elsair et al. 1982
, Herman 1956
). Jolly et al. (1980)
found a reduction in creatinine clearance in patients
with chronic skeletal fluorosis, indicative of glomerular dysfunction.
In our study, we evaluated 45Ca++ uptake by membrane vesicles prepared from the rat kidney cortex following 6 wk of chronic F- ingestion from the drinking water. We selected exposure levels that would produce plasma concentrations of F- attainable by humans from environmental exposures. After chronic exposure to F-, rats were killed, and 45Ca++ uptake was measured. To determine if chronic F- exposure had an effect on Ca++ transport protein expression, independent of direct interaction between F- and Ca++ transport protein activity, the uptake medium for these studies contained no added F-.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Approval of the animal protocol was obtained from the Committee on
Animal Use in Research and Education at the Medical College of Georgia.
Twenty weanling female Sprague-Dawley rats (Harlan, Indianapolis,
IN) were fed a semi-purified rat diet 76A, (Purina Test Diets,
Richmond, IN) (American Institute of Nutrition 1993
) for
the duration of the study (6 wk). This diet was low in F-
(0.9 mg/L), as determined using the Model 9409 ion-specific
electrode (Orion, Boston, MA) and contained 400 mg Ca++/100
g diet, as measured on a Spectra AA atomic absorption spectrometer
(Varian, Sunnyvale, CA). Rats were maintained in a controlled
atmosphere environment at 23°C and 50% relative humidity. Rats were
divided into four groups (see Table 1 for n per group) and were fed ad libitum deionized
water containing F- at 0, 10, 50, or 150 mg/L added as NaF
for 6 wk.
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Animals were anesthetized with ether prior to blood collection and
killed via a pentobarbital overdose without regaining consciousness.
Blood was collected from the right atrium of each rat and lightly
heparinized. Plasma fluoride was analyzed using the ion-specific
electrode (Orion, Model 9409) following overnight diffusion using the
hexamethyldisiloxane-facilitated method of Taves (1968)
as modified
by Whitford (1996a)
. Reference standards for fluoride were prepared by
us using the highest quality ACS-certified reagents available.
Reference standards ranging from 0.25 nmol/L to 100 nmol/L were used
for the fluoride determinations. Plasma ionic calcium was analyzed
using the Microelectronics (Bedford, NH) Model MI-600 Mini-Calcium
Electrode with Model MI-402 Micro-Reference Electrode, following
the manufacturer's instructions. Reference standards for calcium
determinations were prepared by us using the highest quality
ACS-certified reagents available. Reference standards for calcium
were prepared ranging from 10 µmol/L to 100 mmol/L.
45Ca uptake into rat kidney mixed membrane vesicles.
Following their removal, one kidney was frozen for slot blot analysis while the other kidney was used the same day to prepare mixed membrane vesicles without freezing. The cortex was cut from this kidney and homogenized with an Omni 1000 (Omni International, Waterbury, CT) for 1 min in 5 bursts of 12 s each in homogenization buffer containing 100 mmol KCl/L, 5 mmol MgCl2/L, 50 mmolTris HCl/L (pH 7.2), 5 mmol benzamidine/L, 50 mg trypsin inhibitor/L, and 0.5 mmol dithiothreitol/L. Homogenates were centrifuged at 15,600 x g at 4°C for 10 min. The pellet was discarded, and the protein content of the supernatant was determined by the bicinchoninic acid method (Pierce, Rockford, IL). Volumes of supernatant containing 1.8 mg protein each were added to AirfugeTM tubes (Beckman Instruments, Palo Alto, CA), and the volume of each was adjusted to 200 µL. These tubes were centrifuged at 100,000 x g for 1 h at 4°C after which the supernatant was discarded. The mixed membrane pellets were then suspended in 150 µL uptake solution containing 130 mmol KCl/L, 34 mmol K-HEPES/L (pH 7.2), 2 mmol MgCl2/L, 5 mmol K-oxalate/L, 100 µmol CaCl2/L, and 110 µmol M EGTA/L, (free Ca++ = 1.6 µmol/L). To start the uptake reaction, 50 µL of this mixed membrane suspension was added to 500 µL of the same uptake solution, but at 37°C, in a shaker waterbath with 1.5 GBq/L 45CaCl2 and, where appropriate, 5 mmol Tris ATP/L and 2 µmol thapsigargin/L. The uptake reaction was continued for 4 min and stopped by vacuum filtration of the membranes over 0.45 µm HAWP filters (Millipore, Bedford, MA). This time period was used because uptake by these vesicles proceeded in a linear fashion for 4 min. Filters were washed with 9 mL of wash solution containing 150 mmol KCl/L, 20 mmol K-HEPES/L (pH 7.4), and 2 mmol EGTA/L before placing them into scintillation vials for counting. Filters treated in the same way but without kidney membranes were also counted to calculate nonspecific background radioactivity.
Quantitation of plasma membrane and endoplasmic reticulum Ca++-pumps by slot blot analysis.
Quantitation of plasma membrane and endoplasmic reticulum Ca++-pump epitopes in homogenates of rat kidney mixed membranes, involved immobilization of homogenate proteins on nitrocellulose via a Hybri-Slot slot blot apparatus (Bethesda Research Laboratories, Gaithersburg, MD). Samples of homogenate proteins (25 µg) in 50µL of homogenization buffer were loaded into each well of the slot blot apparatus with a vacuum and immobilized on the nitrocellulose. Wells were washed with 500 µL of 50 mmol Tris-HCl/L, pH 7.2. Antigen was visualized on nitrocellulose by the avidin-biotin-peroxidase technique.
Monoclonal antibody 5F10 was obtained from Dr. John Penniston of the Mayo Clinic, Rochester, MN. This antibody reacts with a highly conserved region of all plasma membrane Ca++-pump (PMCA) isoforms. Monoclonal antibody IID8, specific for endoplasmic reticulum Ca++-pump isoform SERCA2 was purchased from BIOMOL, (Plymouth Meeting, PA). Western blot analysis was performed (5F10, 1:1000 or IID8, 1:500) to confirm the specificity of each antibody. The same antibody dilutions were used for both Western blot and slot blot analysis. Diaminobenzidine tetrahydrochloride (DAB) was the substrate for the peroxidase molecule linked to the primary antibodies. The density of brown insoluble precipitate formed by the DAB was analyzed by scanning densitometry.
Data are expressed as means ± SE and were analyzed
for significant differences using ANOVA. Where variances were unequal,
natural log transformation of the data was employed. An 
value of
0.05 was selected a priori. Multiple comparisons versus the control
group were assessed by Dunnett's method.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Body weights and plasma ionic
Ca++ concentrations did not differ among the
groups.
ATP-independent Ca++ uptake and adsorption did
not differ among groups (Fig. 1
). Similarly, in the presence of thapsigargin, a specific inhibitor
of the endoplasmic reticulum Ca++-pump, no
significant differences were observed (Fig. 1)
. ATP-dependent
45Ca++ uptake without
thapsigargin, however, was lower in vesicles prepared from rats with
plasma F- levels of 35 µmol/L than in the
control group (P < 0.05).
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). Thapsigargin-sensitive uptake, representing endoplasmic
reticulum Ca++-pump, also was considered
separately. When the <0.4 µmol/L plasma F-
control group was compared to the 7 µmol/L and 35 µmol/L plasma
F- groups, the lower Ca ++
uptake in the high plasma F- groups was
associated with significantly lower activity of the endoplasmic
reticulum Ca++-pump.
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; P < 0.05). Similar results were obtained for
the endoplasmic reticulum Ca++-pump (Fig 4; P < 0.05).
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). The presence of lightly staining minor bands may represent
breakdown products of the Ca++-pumps. The
possibility that these minor species represent additional proteins with
low affinity binding or additional proteins in low abundance represents
an alternative explanation. However, the overall contribution of these
minor species to the colored reaction product appears very near
background detection levels. This finding suggests that the colored
product used for quantification via slot blot analysis is attributable
to specific antibody binding to Ca++-pump
proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Tissue-to-plasma ratios for kidney, which are well
above unity, represent a major exception to this generalization. The
cortex-to-papilla concentration gradient for fluoride closely resembles
that of sodium and chloride (Whitford and Taves 1973
). Although it was not demonstrated directly, it is also likely the fluoride concentrations among subcellular compartments depend on pH gradients. Thus, it would be expected that intracellular fluoride concentrations would be higher in mitochondria and nuclei in which the pH is higher than in the cytosol. We postulate, therefore, that this concentrating effect of the alkaline nuclear and mitochondrial fluids promotes the elevated fluoride concentrations necessary to affect the regulatory enzymes of the kidney.
A study by Narayanan et al (1991)
showed both inhibitory and
stimulatory effects of F- on the
Ca++-pump of cardiac sarcoplasmic (endoplasmic).
This Ca++-pump, like the
Ca++-pump of the plasma membrane, transports
Ca++ by a series of steps, including the binding
of Ca++ ion; the phosphorylation of the enzyme by
ATP to form a phosphorylated intermediate; and the subsequent
conformational change in the enzyme, which results in the translocation
of the Ca++ across the membrane. Following
translocation of Ca++, the phosphorylated enzyme
intermediate breaks down, releasing Ca++, after
which the enzyme returns to its original conformational state for
another cycle. The reported inhibitory and stimulatory effects of
F- on this process are explained by differing
susceptibilities of the conformational states of the
Ca++-pump to the action of
F-. The susceptibility of the enzyme to
F- inhibition was postulated to occur in the
presence or absence of Ca++, but only for the
dephosphorylated conformation of the enzyme. In these studies,
inhibition occurred when F- was added to the
reaction prior to, but not excluding), the addition of ATP. When
F- was added after the ATP was added, a
stimulatory effect was observed. This action appears similar to the
known inhibitory action of vanadate on the plasma membrane
Ca++ -pump and the
Na+-K+ ATPase
(Cantley et al. 1978
, Inesi et al. 1984
).
The inhibition of these transporters occurs by the interaction of
vanadate with the phosphorylation site of each molecule.
Studies by Holland (1979)
, and Dunapace and Stookey (1988)
, have shown
that F- inhibits both total protein and total
DNA synthesis in cultured mammalian cells. In a recent review of the
genetic toxicity of F-, no apparent direct
mechanism for F- interaction with DNA was
reported, (Zeiger et al. 1993
). A more likely suggestion
is that the inhibition of DNA synthesis may be a secondary effect of
F- interference with protein synthesis or as a
result of the direct interaction of F- with DNA
polymerase or other enzymes associated with DNA synthesis,
(Zeiger et al. 1993
). Several studies have suggested
that F- can interact with divalent cations, such
as Ca++, in the cell, an affect which may disrupt
enzyme systems necessary for DNA or RNA synthesis, or other cellular
processes, (Edwards and Parry 1986
, Harper et al. 1974
, Hellung-Larsen and Klenow 1969
, Holland 1979
, Imai et al. 1983
).
In our study we showed decreases in both the activity (Fig. 2)
and
amount (Figs. 3
and 4)
of plasma membrane and endoplasmic reticulum
Ca++-pumps that corresponded to increases in plasma
F- levels, even when F- was not added to the
assay system. The observation that reductions in Ca++-pump
activity did not correlate directly with changes in the amount of
Ca++-pump protein suggests that additional factors that
affect the activities of the enzymes may be involved. For example,
there may be changes to the Ca++-pump proteins that do not
effect the antigenicity of the proteins. There may also be effects on
the synthesis or degradation of other membrane proteins as well that
affect 45Ca uptake in our system.
Ca++ movement through the kidney tubules is
controlled by Ca++ channels,
Ca++-binding proteins,
Ca++-sequestering organelles,
Na+/Ca++ exchangers, and
Ca++-pumps. Ca++ enters
tubule cells through Ca++ channels down an
electrical and chemical concentration gradient. Cytosolic
Ca++ levels are kept low (near 10
µmol/L) by sequestration in organelles (for example, through
the action of the endoplasmic reticulum
Ca++-pump) or through binding to
calcium-binding proteins. Ca++ efflux from
cells occurs by means of a
Na+/Ca++ exchanger or a
plasma membrane Ca++/Mg++
ATPase Ca++-pump. Whereas both of these
mechanisms function in the nephron of rats, the physiological role of
the Na+/Ca++ exchanger is
unclear. Studies by Gmaj and Murer (1988)
suggest that under normal
physiological conditions, the
Na+/Ca++ exchanger may
contribute only 10% as much to Ca++ efflux as
the plasma membrane Ca++-pump. Our studies used
Na+-free incubation conditions to eliminate the
role of the Na+/Ca++
exchanger in vesicle uptake.
In our study, a decrease in 45Ca++ uptake was observed in kidney membranes isolated from rats after chronic F- ingestion. The novel and important aspect of our study was that 45Ca++ uptake was measured in uptake medium in the absence of added F-. The decrease in Ca++ transport activity in these membranes was due to a decreased production of the enzyme by the kidney cells, an increased turnover of the enzymes, or an increased breakdown of the enzymes by the cells. Stated differently, F- appears to have affected the expression of the enzymes independent of any effect that F- may have directly on the enzyme activity. Our study provides the first evidence that one of the effects of long-term F- exposure is a change in expression of the plasma membrane and endoplasmic reticulum Ca++-pumps in the kidney.
In summary, we provided rats with fluoride in their drinking water, which produced graded, plasma fluoride concentrations that occur in humans. Our studies showed that chronic high fluoride ingestion decreases the rate of Ca++ transport across renal tubule endoplasmic reticulum and plasma membranes, and reduced the amount of ER and PM Ca++-pump protein present in the kidney membranes. We conclude that chronic high fluoride ingestion may decrease the expression, increase the breakdown, or increase the rate of turnover of plasma membrane and endoplasmic reticulum Ca++-pump proteins and possibly other enzymes as well. The observed decreases in the rate of Ca++ transport and associated decreases in plasma membrane and endoplasmic reticulum Ca++-pump expression could affect in vivo Ca++ homeostasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: DAB, diaminobenzidine
tetrahydrochloride; F-, fluoride; PMCA, plasma membrane
Ca++-pump.
Manuscript received June 15, 1998. Initial review completed July 14, 1998. Revision accepted February 5, 1999.
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