1 Endocrine Research Unit, Department of Medicine, Veterans Affairs Medical Center, University of California, San Francisco, California 04121; 3 Institution of Molecular Pharmacology and Biophysics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267; 2 Division of Clinical Pharmacology, First Department of Internal Medicine, Imre Haynal University of Health, H-1135 Budapest, Hungary
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ABSTRACT |
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Parathyroid cells express
Ca2+-conducting currents that are activated by raising the
extracellular Ca2+ concentration
([Ca2+]o). We investigated the sensitivity of
these currents to dihydropyridines, the expression of voltage-dependent
Ca2+ channel (VDCC) subunits, and the effects of
dihydropyridines on the intracellular free [Ca2+]
([Ca2+]i) and secretion in these cells.
Dihydropyridine channel antagonists dose dependently suppressed
Ca2+-conducting currents, and agonists partially reversed
the inhibitory effects of the antagonists in these cells. From a bovine
parathyroid cDNA library, we isolated cDNA fragments encoding parts of
an 1S- and a
3-subunit of L-type
Ca2+ channels. The
1S-subunit cDNA from the
parathyroid represents an alternatively spliced variant lacking exon 29 of the corresponding gene. Northern blot analysis and
immunocytochemistry confirmed the presence of transcripts and proteins
for
1- and
3-subunits in the parathyroid
gland. The addition of dihydropyridines had no significant effects on
high [Ca2+]o-induced changes in
[Ca2+]i and parathyroid hormone (PTH)
release. Thus our studies indicate that parathyroid cells express
alternatively spliced L-type Ca2+ channel subunits, which
do not modulate acute intracellular Ca2+ responses or
changes in PTH release.
intracellular Ca2+ mobilization; Ca2+ receptor; Ca2+ sensing
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INTRODUCTION |
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CHANGES IN THE EXTRACELLULAR Ca2+ concentration ([Ca2+]o) regulate parathyroid hormone (PTH) secretion through the coupling of Ca2+-sensing receptors (CaRs) to signaling pathways (5). With Ca2+ binding and CaR activation, phospholipase C activity increases, cAMP production decreases, and intracellular Ca2+ concentrations ([Ca2+]i) rise (4-6, 9, 11, 29). Studies with pharmacological agents that raise [Ca2+]i suggest that incremental changes in this mediator are linked to the inhibition of PTH secretion (27, 30). High [Ca2+]o-induced increases in 1,4,5-inositol trisphosphate are temporally linked to the initial rapid phase of Ca2+ mobilization from intracellular stores (29), whereas Ca2+ influx across the membrane is probably responsible for sustained changes in [Ca2+]i (3).
Studies by Fitzpatrick and colleagues (16, 18, 26) have suggested a role for L-type Ca2+ channels in regulating intracellular Ca2+ mobilization and PTH release. Other groups, however, found no effects of dihydropyridines on the same parameters (23). Expression of classic L-type channels in parathyroid cells is further contested by the observation that membrane depolarization, which activates L-type Ca2+ channels, has no significant effect on [Ca2+]i or parathyroid function (28, 37). These aspects of parathyroid cell signal transduction remain controversial. The current studies were undertaken to address whether parathyroid cells express L-type Ca2+ channels and whether these channels participate in high [Ca2+]o-regulated cellular functions.
On the basis of electrophysiological and pharmacological criteria,
voltage-dependent Ca2+ channels (VDCCs) are
classified into L-, N-, T-, P/Q-, and R-types (1). Each of
these channels consists of a pore-forming 1-subunit and
accessory proteins, including
2/
-,
-, and
-subunits (7, 8, 32, 36). The structure of the
1-subunit determines ion selectivity, voltage
sensitivity, and binding specificity to its ligands (7, 33, 36,
38). L-type VDCCs are characterized by their sensitivity to
changes in membrane potential and high affinities for
1,4-dihydropyridines, phenylalkylamines, and benzothiazepines (1,
33, 36). Three major subtypes of L-type
1-subunits have been described, including: 1)
the
1C in heart, smooth muscle, and neurons;
2) the
1S in skeletal muscle; and
3) the
1D in neuroendocrine cells (1,
33, 35). Each
1-subunit contains four
membrane-associated motifs (I-IV), and each motif is comprised of six
membrane-spanning domains (S1-S6). Recent studies have identified the
dihydropyridine binding sites in the S5 and S6 domains of motif III
(III-S5 and III-S6) and the S6 domain of motif IV (IV-S6) of L-type
1-subunits (8, 33). The sensors of membrane
potential are also localized to the S6 domain in each motif (I-IV)
(2, 36). The genomic structure of L-type
1-subunits is relatively complex. The human skeletal
1S-subunit gene, for example, spans 90 kb and consists
of 44 exons (20). Alternatively spliced transcripts of
this gene have been identified and possibly contribute to the molecular
and functional diversity of L-type
1-subunits in
different tissues (15, 25).
We previously identified and characterized nifedipine-sensitive
cation-selective currents in parathyroid cells, whose activity increased with rising [Ca2+]o
(9). Although these currents can conduct Ca2+,
they are not voltage gated like other dihydropyridine-sensitive L-type
channels (9). These findings suggested that the channels that bind nifedipine and conduct Ca2+ currents in
parathyroid cells may differ from classic L-type channels. To define
further the pharmacology and molecular identity of high
[Ca2+]o-induced, nifedipine-sensitive cation
currents in these cells, we examined the effects of different
dihydropyridine agonists and antagonists on these currents and isolated
cDNA fragments encoding L-type channel subunits. We found that the high
[Ca2+]o-activated Ca2+ currents
were modulated in a dose-dependent manner by (+)- and ()202-791
and R- and S-BAY K 8644. Clones isolated from a bovine parathyroid cDNA
library showed substantial homology to human
1S- and
3-subunits of L-type Ca2+ channels. The
expression of RNA transcripts and protein of L-type channel subunits in
parathyroid cells was further confirmed by Northern analysis and
immunocytochemistry. We further found that dihydropyridines had no
significant effects on high [Ca2+]o-induced
increases in [Ca2+]i and PTH release,
suggesting that L-type Ca2+ channels in parathyroid cells
do not participate in the immediate responses to the changes in
[Ca2+]o.
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MATERIALS AND METHODS |
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Materials
R- and S-BAY K 8644 were purchased from Calbiochem (La Jolla, CA). (+)- and (Preparation of Parathyroid Cells
Bovine parathyroid cells were isolated after collagenase and DNase digestion of parathyroid gland fragments, as previously described (9). For electrophysiological studies, isolated cells were plated on no. 1 round coverglasses and incubated for 30 min at 37°C before recording (9). For measurements of [Ca2+]i, cells were cultured on no. 1 round coverglasses for 12-18 h in MEM with FCS (2%) and penicillin/streptomycin (100 U/ml).Whole Cell Recording
Whole cell voltage clamping was performed using glass pipettes with an electrical resistance of 1-4 M
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Electrode solutions. Recordings were performed with a whole cell electrode solution (WCES) containing (in mM): 140 Cs-MES, 5 MgCl2, 10 EGTA, 10 HEPES (pH 7.4), 4 MgATP, 0.3 GTP, and a nucleotide-regenerating system (NRS: 14 mM phosphocreatine and 50 U/ml creatine phosphokinase) (9).
Bath solutions and extracellular bath perfusion.
All bath solutions (BS) contained 10 mM HEPES (pH 7.4) and 10 mM
tetraethylammonium (TEA+) to block endogenous
K+ currents (9). Various [Ca2+]
in the BS (0.7-90 mM) were achieved by the addition of Ca acetate. Acetate was used as the anion charge-carrier to minimize the activity of endogenous Cl currents (9). Osmolarity of
the BS was adjusted to
330 mosM/l with sucrose as needed. Each BS is
specified by the concentration of its major cation species as follows
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Screening of PTH cDNA Library
A bovine parathyroidRT-PCR
Total and poly A+ RNA were isolated from newborn calf parathyroid glands and human parathyroid adenomas with an RNA Stat-60 kit (10). The primers used for bovine (upper: 5'-ACAACCAGTCAGAGCAAATG-3'; lower: 5'-ACACGGAACAGGCGG AAGAA-3') and human (upper: 5'-TGGCCTTCACTATCATCTTC-3'; lower: 5'-GGGTTCGCACTCCTTCTG-3')Immunocytochemistry
Immunocytochemistry of bovine parathyroid sections was performed as described (10) using the anti-panMeasurement of [Ca2+]i and PTH Release
[Ca2+]i was determined using an InCyt Im2 imaging system (Intracellular Imaging, Cincinnati, OH) with a ×40 Nikon Fluor objective. Briefly, cells were loaded with fura 2-AM (3 µM) in buffer A [20 mM HEPES (pH 7.4), 120 mM NaCl 5 mM KCl, 1 mM MgCl2, 1 mg/ml pyruvate, 1 mg/ml glucose, and 1.0 mM CaCl2] at 37°C for 30-40 min. After three washes with buffer A, cells were incubated at 37°C for 15-30 min before recording. Fluorescent emission (510 nm) was detected by a COHU high-performance charge-coupled device camera (COHU, San Diego, CA), digitized, and stored in a microcomputer. The 340/380 excitation ratio (R340/380) of emitted fluorescence was calculated.PTH release was measured from cells treated with vehicle (0.1%
ethanol) or dihydropyridines (106 M) for 30 min at 37°C
(31).
Statistics
Data, normalized to baseline activity in individual experiments, were combined and reported as means ± SE. Statistical significance was determined by ANOVA with an f-test using Microsoft Excel computer software (Microsoft, Seattle, WA). ![]() |
RESULTS |
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Dihydropyridine Agonists and Antagonists Modulate Ca2+-Conducting Currents
In previous studies, we characterized two types of Ca2+-conducting currents in bovine parathyroid cells. Type 1 Ca2+ currents increased with rising [Ca2+]o and were blockable by Cd2+, La3+, Gd3+, and nifedipine. Type 2 currents, which were insensitive to Cd2+ and nifedipine, could be blocked by La3+ and Gd3+ (9, 10). To determine whether type 1 currents were sensitive to other dihydropyridines, we tested the effects of (+)- and (The effects of ()202-791 on the Ca2+-conducting
currents could be partially reversed by its isomeric counterpart
(+)202-791, as demonstrated in Fig.
2. In a representative experiment,
addition of (
)202-791 (10
8 M) suppressed
Im and Gm by
60%
(Fig. 2A, i and ii, and B).
Subsequent perfusion of the cell with (+)202-791 (3 × 10
6 M) partially restored Im (Fig.
2A, iii) and Gm (Fig.
2B). Recovery of Im and
Gm was not due to damage to the membrane-pipette
seal, because both parameters could be suppressed by Cd2+,
a blocker of type 1 currents (Fig. 2A, iv, and
B), and La3+ (Fig. 2B).
R- and S-BAY K 8644 also modulated type 1 currents in parathyroid
cells. As shown in Fig. 3, the
Im and Gm induced by high [Ca2+]o (i and ii) were
suppressed by R-BAY K 8644 (106 M) (iii). The
inhibitory effects of R-BAY K 8644 were reversed by addition of S-BAY K
8644 (10
5 M), an L-type channel agonist (Fig.
3iv). Again, the recovery of Im and
Gm by S-BAY K 8644 was not the result of leakage
of the membrane seal, because these parameters could be further
suppressed by nifedipine (10
5 M; Fig. 3v) and
Gd3+ (3 × 10
3 M; Fig. 3 vi).
These studies strongly suggest that dihydropyridine-sensitive L-type
channels are present in parathyroid cells. The insensitivity of
Ca2+-conducting currents to membrane potential, however,
suggested that the channel subunits interacting with dihydropyridines
and responsible for conducting Ca2+ currents in these cells
may differ from classic L-type VDCCs.
Cloning of Parathyroid Channel Subunit cDNAs
To determine the identity of putative L-type channel subunits in parathyroid cells, we screened a parathyroid cDNA library for the presence of L-type
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The amino acid sequences derived from the PT 1-1
cDNA clone revealed the presence of putative dihydropyridine-binding
domains (Fig. 4A, III-S5, III-S6, and IV-S6 domains) and
membrane potential sensors (III-S6 and IV-S6 domains). One striking
difference between the PT
1-1 clone and the human
skeletal
1S homologue is a deletion of 19 amino acids
within the linkers between the IV-S3 and IV-S4 domains. These amino
acids are encoded by 57 nucleotides, consisting of exon 29 of the
1S-subunit gene (20). To confirm whether such a deletion resulted from alternative transcript splicing or was a
cloning artifact, we performed RT-PCR using poly A+ RNA
isolated from bovine parathyroid and human parathyroid adenoma tissue.
In four experiments from three separate RNA preparations, we
consistently amplified
1-subunit cDNA lacking exon 29 from bovine (Fig. 5A) and
human (Fig. 5B) parathyroid tissues. These findings
indicated that the parathyroid gland predominantly expresses the
alternatively spliced variant of the L-type
1-subunit.
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RNA and Protein Expression of the L-type 1- and
3-Subunit in PTH
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To assess the protein expression of 1-subunits in
parathyroid cells, we performed immunocytochemistry using an
anti-pan-
1 antiserum raised against an epitope that is
conserved among
1C-,
1S-, and
1D-subunits. Brown DAB staining for the
1-subunit was localized to parathyroid cells and smooth
muscle cells of nearby arterioles and venules (Fig.
7, a and b).
Preincubation of antibodies with pan-
1 peptide prevented
staining, indicating specificity of the antibody (Fig. 7c).
These findings further confirmed the presence of L-type
Ca2+ channels in parathyroid cells.
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Effects of Dihydropyridines on the Intracellular Ca2+ Concentration and PTH Secretion in Parathyroid Cells
To test whether the dihydropyridine-sensitive type 1 current contributes to the high [Ca2+]o-induced increases in [Ca2+]i in parathyroid cells, we measured [Ca2+]i by microfluorimetry in the presence and absence of (
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We next tested the effects of dihydropyridines on PTH release. Whereas
high [Ca2+]o suppressed PTH release, the
addition of (+)202-791 or R-BAY K 8644 did not affect PTH release
at any concentration tested (109 to 10
6 M)
(Table 1 and data not shown). In
addition, (
)202-791, S-BAY K 8644, and nifedipine did not alter
PTH release (data not shown), suggesting that L-type Ca2+
channels may not play a role in modulating PTH secretion.
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DISCUSSION |
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Changes in [Ca2+]o regulate PTH secretion by interacting with CaRs in the membranes of parathyroid cells (5). High [Ca2+]o inhibits PTH release, whereas maximal secretion occurs at low [Ca2+]o. Studies with ionomycin and thapsigargin, which mobilize intracellular Ca2+, suggest that increasing [Ca2+]i is an important step in suppressing PTH release (27, 30). In parathyroid cells, initial increases in [Ca2+]i that result from raising [Ca2+]o are thought to be mediated by Ca2+ released from intracellular stores (24, 29). The sustained elevation of [Ca2+]i, on the other hand, requires Ca2+ entry (19, 37). In other cell systems, Ca2+ influx across the membrane involves the opening of Ca2+ channels. In the present study, we showed that the channel blockers La3+ and Gd3+ could block high [Ca2+]o-induced Ca2+-conducting currents and sustained Ca2+ mobilization caused by raising [Ca2+]o. These observations suggest that Ca2+ entry in parathyroid cells is also mediated by ion channels.
Several studies have investigated the nature of the channels
responsible for high [Ca2+]o-induced
Ca2+ influx in parathyroid cells. Certain observations
suggested an L-type Ca2+ channel as a potential candidate
(26). Fitzpatrick et al. (17) showed that the
L-type channel agonist (+)202-791 suppressed and the antagonist
()202-791 enhanced PTH secretion in dispersed adult bovine
parathyroid cells. This group (Fitzpatrick et al., 18) also detected by
use of an L-type channel antiserum a protein in parathyroid cell
lysates with a size (
150 kDa) comparable to the skeletal
1s-subunit. Furthermore, incubation of cells with this
same antiserum reduced PTH secretion, suggesting that this antiserum
could bind to and activate the putative Ca2+ channel. In
support of this possibility, increased isotopic Ca2+ flux
was also demonstrated after preincubation of cells with this antibody
(18). Other evidence linking L-type channels to parathyroid function was reported by Cooper et al. (13),
who showed that BAY K 8644 increased [Ca2+]i
and Ca2+ influx in parathyroid cells. In the current
investigation, we recorded membrane currents that were sensitive to
dihydropyridines in isolated parathyroid cells. Our immunocytochemistry
and Northern blotting provided evidence that protein and mRNA encoding
putative L-type
1- and
-subunits were expressed in
these cells. Finally, cloning and RT-PCR data confirmed the presence of
an alternatively spliced skeletal
1S-subunit in these
cells. Taken together, our observations and those of other
investigators support the idea that parathyroid cells express a
skeletal muscle-like isoform of the L-type Ca2+ channel and
that these channels might mediate changes in
[Ca2+]i and PTH release.
Our functional studies, however, yielded somewhat unexpected results, given the work of others summarized above and our own electrophysiology experiments. We were unable to demonstrate statistically significant effects of dihydropyridines on high [Ca2+]o-induced increases in [Ca2+]i in parathyroid cells or PTH release with a variety of acute and short-term incubation protocols. These observations confirm those of Muff et al. (23), who found no effects of (±)202-791 on PTH release or [Ca2+]i.
At present, we have no explanation for the inconsistencies in studies
with dihydropyridines among different laboratories. They may be
attributed to differences in methods and reagents. Variations could
also be due to the ages of the animals from which parathyroid tissues
were obtained. Our studies and those of Muff et al. (23)
employed glands from newborn calves, whereas Fitzpatrick and coworkers
(16-18) used adult bovine parathyroid glands. The expression of L-type 1-subunits may be developmentally
regulated in certain tissues. For instance,
1-subunit
expression increases in the adult brain compared with embryonic brain
(15). Whether the expression of L-type Ca2+
channel subunits increases with age in the parathyroid and whether these subunits become functional in mediating Ca2+ influx
only in cells from adult glands are possibilities that will require
further investigation.
The partial 1-subunit cDNA we cloned from the
parathyroid appears to be an alternatively spliced product of the
skeletal muscle
1s-subunit gene (20). This
cDNA lacks the 57 nucleotides that correspond to exon 29 of the human
homolog of this gene and that encode 19 amino acids within the linker
between IV-S3 and IV-S4 domains. Such a deletion would shorten this
extracellular loop from 32 to 13 amino acids. Similar spliced products
have been previously identified in skeletal muscle-like BC3H1 cells, mouse ovary, rabbit intestinal smooth muscle, and rat brain (15, 25). Our RT-PCR experiments detected only cDNAs lacking exon 29 in bovine and human parathyroid tissues, suggesting that the spliced
variant is the predominant one expressed in the parathyroid. The
significance of such a structural modification on channel function in
any system, however, remains unclear.
Parathyroid cells are nonexcitable. Membrane depolarization by high K+ does not induce Ca2+ influx (28, 37). The lack of a response to depolarizing concentrations of K+ could be due to the lack of expression of classic VDCCs (i.e., dihydropyridine-responsive channels) or the expression of channels with altered voltage-sensing properties. Our electrophysiological data support the latter possibility (9). Using a traditional whole cell patch-clamp configuration, we recorded dihydropyridine-sensitive membrane currents in parathyroid cells that were voltage independent. Their affinity for dihydropyridines, however, was comparable to that of classic VDCCs. The nifedipine-sensitive currents in parathyroid cells were cation nonselective, in contrast to typical VDCCs in excitable cells, which exhibit significant selectivity for Ca2+. We did not record other voltage-sensitive Ca2+-conducting currents with the same protocols. It is therefore likely that the channels that bind dihydropyridines in parathyroid cells are not sensitive to changes in membrane potential.
Clearly, because their electrophysiological properties differ, the
molecular characteristics of the responsible channel subunits in
parathyroid cells must diverge from classic L-type VDCCs. By surveying
the deduced partial amino acid sequence of the parathyroid 1-subunit cDNA, we were able to identify the putative
dihydropyridine-binding domains (i.e., III-S5, III-S6, and IV-S6) and
membrane potential-sensing domains (i.e., III-S4 and IV-S4). The
expression of dihydropyridine-binding regions supports data showing
that these drugs bind to parathyroid cell membranes (21).
The presence of voltage-sensing domains may, however, contradict our
electrophysiological results, because the dihydropyridine-blockable
currents we recorded were not classically voltage gated. Because the
3.3-kb parathyroid
1-subunit cDNA we cloned represents
only
50% of the full-length transcript, it is likely that
modifications in the as-yet-unknown portions of the cDNA could
potentially alter the ability of the channels to sense changes in
membrane potential. The confirmation of this possibility requires the
cloning of the full-length parathyroid
1-subunit cDNA.
Alternatively, the protein product of the full
1-subunit, whose partial clone we identified, may not
mediate the voltage-insensitive currents in these cells. Other
as-yet-unidentified channel subunits may be responsible for conducting
these currents, or other channel regulators or subunits may modify the
voltage dependency of the multisubunit channel complex in parathyroid cells.
A major unresolved issue inherent in these data is the difference between the electrophysiological and microfluorimetric findings. Electrophysiological recordings clearly show that dihydropyridine agonists and antagonists activate or block, respectively, cation currents in parathyroid cells that are responsive to changes in [Ca2+]o and that can be carried by Ca2+ (9). Yet, surprisingly, these same agents did not detectably alter [Ca2+]i, either in the basal state or in response to raising [Ca2+]0 under a variety of short-term protocols. Our intracellular Ca2+ determinations were done on groups of cells, from which single cells responding with even a 20% change in [Ca2+]i could have been reliably detected. No significant differences in [Ca2+]i over as long as 60 min were seen. Although we cannot explain why dihydropyridines had no effect on [Ca2+]i, we think that differences in methodology may be important to consider. 1) Patch-clamping is a more sensitive method for recording ion channel activity than is microfluorimetry. Because we performed electrophysiological recordings in the presence of high [Ca2+]o (90 mM), which greatly enhanced type 1 currents, we could demonstrate clearly a blockade of the currents by dihydropyridines. In microfluorimetry, Ca2+ fluxes induced by smaller but more physiological increments of [Ca2+]o (from 0.5 to 2.5 or 5.0 mM) could have occurred. Perhaps, however, the changes in [Ca2+]i were too small to alter [Ca2+]i over the entire cytosol. Other aspects of Ca2+ mobilization activated by CaRs, such as the release of intracellular Ca2+ and Ca2+ influx via dihydropyridine-insensitive channels, could also have masked subtle changes in [Ca2+]i brought about by dihydropyridines in single-cell microfluorimetry experiments. 2) With intact cells, the recording conditions for microfluorimetry, Ca2+ may not be the predominant ion carried by these dihydropyridine-sensitive currents. Hence, [Ca2+]i may not change with these drugs. In support of this possibility, our previous studies clearly show that these currents can be conducted by divalent as well as monovalent cations. Given the lack of available parathyroid-derived cDNAs for the other channel subunits, it is not possible to address definitively the basis for different results from electrophysiological vs. microfluorimetric studies.
The differences between the two types of data emphasize the importance of using electrophysiological approaches as a guide for hypothesis generation and protocol development. Ultimately, physiological principles must always be tested in intact cells, and eventually in animals, to assure that they are valid with respect to physiological events in vivo, including hormone secretion and second-messenger generation. A more complete understanding of the functions served by dihydropyridine channel subunits in nonexcitable tissues will eventually require more complete information on the subunit structure and molecular properties of the ion channels expressed in the parathyroid.
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ACKNOWLEDGEMENTS |
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We appreciate the assistance of Dr. Orlo Clark in the procurement of human parathyroid tissue and the support of Vivian Wu in the preparation of this manuscript.
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FOOTNOTES |
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During these studies, Dr. Shoback was supported by a Merit Review from the Department of Veterans Affairs, the Northern California Arthritis Foundation, and National Institutes of Health (NIH) Grant DK-43400, and Dr. Schwartz was supported by NIH Grant HL-43231.
Address for reprint requests and other correspondence: D. Shoback, 111N, Endocrine Research Unit, VA Medical Center, 4150 Clement St., San Francisco, CA 94121 (E-mail: dolores{at}itsa.ucsf.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 July 2000; accepted in final form 7 March 2001.
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