Parathyroid cells express dihydropyridine-sensitive cation currents and L-type calcium channel subunits

Wenhan Chang1, Stacy A. Pratt1, Tsui-Hua Chen1, Chia-Ling Tu1, Gabor Mikala2, Arnold Schwartz3, and Dolores Shoback

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1S- and a beta 3-subunit of L-type Ca2+ channels. The alpha 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 alpha 1- and beta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1-subunit and accessory proteins, including alpha 2/delta -, beta -, and gamma -subunits (7, 8, 32, 36). The structure of the alpha 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 alpha 1-subunits have been described, including: 1) the alpha 1C in heart, smooth muscle, and neurons; 2) the alpha 1S in skeletal muscle; and 3) the alpha 1D in neuroendocrine cells (1, 33, 35). Each alpha 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 alpha 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 alpha 1-subunits is relatively complex. The human skeletal alpha 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 alpha 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 alpha 1S- and beta 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.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Materials

R- and S-BAY K 8644 were purchased from Calbiochem (La Jolla, CA). (+)- and (-)202-791 were obtained from Calbiochem and Novartis Pharma (Basel, Switzerland). Media were prepared by the Cell Culture Facility of the University of California, San Francisco. Antibodies against the alpha 1-subunit of L-type Ca2+ channels (anti-pan-alpha 1) were raised against a peptide (pan-alpha 1, amino acid sequence: DNFDYLTRDWSILGPHHLD) within an intracellular domain between motif IV and the COOH terminus and were affinity-purified by Alomone Labs (Jerusalem, Israel). This epitope is highly conserved among L-type alpha 1-subunits, including alpha 1C, alpha 1D, and alpha 1S. Donkey anti-rabbit IgG antibodies were purchased from Amersham Life Science (Arlington Heights, IL). Fura 2-AM was obtained from Molecular Probes (Eugene, OR). All other channel blockers, salts, and chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. All sequencing was done by the Biomolecular Resources Center (University of California, San Francisco). Human parathyroid glands were obtained at the time of surgery for primary hyperparathyroidism according to protocols approved by the Committee on Human Research at the University of California, San Francisco.

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 MOmega , as previously described (9). Membrane potential (Vm) was controlled, and membrane current (Im) was detected by an Axo-Patch amplifier (Axon Instruments, Foster City, CA). Channel activity was assessed by calculating the membrane conductance (Gm) derived from the slope of the Im-Vm plots. Im-Vm plots were created using the following voltage-clamping protocol (Fig. 1, i). Cells were held at -60 mV; then a series of 150-ms test voltage pulses were applied at 2-s intervals in increments of 20 mV from -100 to +120 mV. The current traces were recorded from 20 ms before to 25 ms after each applied voltage pulse. The downward and upward deflections represent the inward and outward currents, respectively. Arrows in Figs. 1-3 represent zero current level. Membrane currents used for making Im-Vm plots are the arithmetic means of the currents recorded during the voltage pulses. Representative experiments are shown in Figs. 1-3, and all experiments were performed on >= 3 cells at room temperature, unless otherwise specified.


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Fig. 1.   Inhibition of the Ca2+-conducting currents in bovine parathyroid cells by dihydropyridine (-)202-791. A: whole cell patch-clamp recordings were made (as described in MATERIALS AND METHODS) from a cell perfused with 0.7 Ca/10 TEA acetate bath solution (BS, ii) and 90 Ca/10 TEA acetate BS (iii-viii) in either the absence (iii) or presence of (-)202-791 (iv-viii, 10-10 to 0-4 M) and La3+ (viii, 3 × 10-3 M). Panel i shows voltage-clamping protocol. B: membrane conductance (Gm) derived from the slopes of membrane current (Im- membrane potential (Vm) plots shown in A. Arrows, 0 current level.



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Fig. 2.   The dihydropyridine agonist (+)202-791 partially reversed the (-)202-791-induced inhibition of Ca2+-conducting currents in bovine parathyroid cells. A: whole cell patch-clamp recordings were made as described in MATERIALS AND METHODS in 90 Ca/10 TEA acetate BS in either the absence (i) or presence of (-)202-791 (ii-iv, 10-8 M), (+)202-791 (iii-iv, 3 × 10-6 M), or Cd2+ (iv, 3 × 10-3 M). B: Gm derived from the slopes of Im-Vm plots shown in A. Pipette solution: whole cell electrode solution (WCES).



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Fig. 3.   Effects of R- and S-BAY K 8644 on the Ca2+-conducting currents in bovine parathyroid cells. A: whole cell patch-clamp recordings were made in either 0.7 Ca/10 TEA (i) or 90 Ca/10 TEA acetate BS (ii-vi) in the absence (i and ii) or the presence of R-BAY K 8644 (iii-iv, 10-6 M), S-BAY K 8644 (iv, 10-5 M), nifedipine (Nif) (v, 10-5 M), or Gd3+ (vi, 3 × 10-3 M).

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 approx 330 mosM/l with sucrose as needed. Each BS is specified by the concentration of its major cation species as follows
90 Ca<IT>/10 </IT>TEA acetate BS<IT>=</IT>(in mM)<IT> 90 </IT>Ca<SUP><IT>2+</IT></SUP><IT>, 10 </IT>TEA<SUP><IT>+</IT></SUP><IT>,</IT>

<IT> 190 </IT>acetate<SUP><IT>−</IT></SUP><IT>, 10 </IT>HEPES (pH<IT> 7.4</IT>)

0.7 Ca<IT>/10 </IT>TEA acetate BS<IT>=</IT>(in mM)<IT> 0.7 </IT>Ca<SUP><IT>2+</IT></SUP><IT>, 10 </IT>TEA<SUP><IT>+</IT></SUP><IT>,</IT>

<IT> 11.4 </IT>acetate<SUP><IT>−</IT></SUP><IT>, 10 </IT>HEPES (pH<IT> 7.4</IT>)<IT>, </IT>and<IT> 267 </IT>sucrose
Recording and cell perfusion were done in a Lucite perfusion chamber with a volume of 0.8 ml, as previously described (9). Channel antagonists and agonists were premixed with appropriate BS and then delivered to the recording chamber. All recordings were initiated >= 10 min after delivery of a given BS.

Screening of PTH cDNA Library

A bovine parathyroid lambda  ZAP II cDNA library, prepared from newborn calf parathyroid poly A+ RNA (Stratagene, La Jolla, CA), was screened according to the manufacturer's instructions. Briefly, lambda  cDNA was first mixed with and allowed to infect XL1-Blue Escherichia coli. Infected cells were then plated on 150-mm agar plates (50,000 pfu/plate) and grown at 37°C for 8-12 h until plaques were clearly identified. Nitrocellulose membrane replicas of the plates were fixed and probed for the gene of interest by use of standard DNA-DNA hybridization with probes prepared from a rat aorta alpha 1-subunit [VASM alpha -1.3 (22)] and a human beta 3-subunit cDNA (12). Positive plaques were isolated, and pBluescript SK(-) phagemids containing inserts of interest were excised from lambda  ZAP II according to the manufacturer's instructions. Excised pBluescript SK(-) phagemids were amplified, and inserts were sequenced. Approximately 106 pfus were screened with the alpha -1.3 and beta 3 cDNA probes.

RT-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') alpha 1S-subunits were derived from the sequences of the bovine parathyroid cDNAs identified in this study (PT alpha 1-1) and the human skeletal muscle homologue (20) (GenBank: L33798). PCR was performed on the first strand cDNA reverse transcribed from bovine or human parathyroid poly A+ RNA (approx 0.5 µg), as described. The following reaction temperatures and durations were used: 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, each for 30 cycles. The reaction ended with a 7-min extension at 72°C. PCR products were electrophoresed on agarose gels, visualized by ethidium bromide staining, subcloned into pT-Adv (Clontech Laboratories, Palo Alto, CA) or PCR II-TOPO (Invitrogen, Carlsbad, CA) vectors by use of TA cloning methods according to the manufacturer's instructions, and sequenced.

Immunocytochemistry

Immunocytochemistry of bovine parathyroid sections was performed as described (10) using the anti-pan alpha 1 antiserum (4 × 10-7 M). To assess specificity, sections were treated with either antiserum preincubated with excess peptide (2.4 × 10-5 M) or nonimmune rabbit IgG. After diaminobenzene (DAB) staining, cells or sections were counterstained with aqueous hematoxylin (10). Experiments were repeated four times on tissue sections from two animals.

Measurement 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 (10-6 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 (-)202-791 and R- and S-BAY K 8644 on high [Ca2+]o-induced Ca2+ currents. As shown in Fig. 1, the dihydropyridine antagonist (-1)202-791 suppressed Im and Gm in a dose-dependent manner (10-10 to 3 × 10-7 M), with an IC50 of approx 10-9 M. In the presence of a maximum dose of (-)202-791, the residual type 2 currents that accounted for approx 50% of total currents were further blocked by La3+ (3 × 10-3 M). The reversal potential (Vr), derived from Im-Vm plots, did not shift significantly with (-)202-791 (data not shown), suggesting that this blocker did not affect the ion selectivity of these currents. These results are compatible with our previous findings for nifedipine and indicate that dihydropyridines affect only type 1 currents.

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 approx 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 (10-6 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 alpha 1- and beta 3-subunit cDNAs. We isolated three partial alpha 1-subunit cDNAs with sizes of approx 3.3 kb (PTalpha 1-1), approx 0.8 kb (PTalpha 1-2), and approx 0.3 kb (PTalpha 1-3). Sequencing revealed that clones PTalpha 1-2 and PTalpha 1-3 were part of the PTalpha 1-1 clone (data not shown). The nucleotide and predicted amino acid sequences of PTalpha 1-1 were 86 and 88% identical to those of the human skeletal muscle alpha 1S-subunit, respectively (Fig. 4A and data not shown). Using the same library, we also isolated a partial beta 3-subunit cDNA (approx 488 bp) whose predicted amino acid sequence was 99% identical to the human VDCC beta 3-subunit (Fig. 4B).


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Fig. 4.   Deduced amino acid sequences of alpha 1 (PTalpha 1-1)- and beta 3 (PTbeta 3)-subunit cDNAs isolated from a parathyroid cDNA library as described in MATERIALS AND METHODS. A: sequence alignment of the PTalpha 1-1 subunit (bPT, top row) and human skeletal alpha 1S-subunit (h-SkM, bottom row); Genebank accession number: L33798. B: sequence alignment of the PTbeta 3-subunit (b-PT, top row) and human beta 3-subunit (h-Emb, bottom row); Genebank accession number: X76555.

The amino acid sequences derived from the PT alpha 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 PTalpha 1-1 clone and the human skeletal alpha 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 alpha 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 alpha 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 alpha 1-subunit.


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Fig. 5.   RT-PCR amplification of the alpha 1-subunit cDNA that contains an in-frame deletion of 57-bp nucleotides from parathyroid tissues. A: ethidium bromide-stained gel containing RT-PCR products amplified from bovine (b) and human (h) parathyroid (PT) RNA. B and C: nucleotide sequences of the RT-PCR products from bovine (top line, B) and human (top line, C) parathyroid were compared with the corresponding region of the human skeletal muscle alpha 1S-unit cDNA (bottom lines, B and C). Partial sequences for the primers used in the RT-PCR are underlined.

RNA and Protein Expression of the L-type alpha 1- and beta 3-Subunit in PTH

To examine the sizes of the full-length transcripts for the parathyroid alpha 1- and beta 3-subunits, we performed Northern blot analysis. With probes made from the PTalpha 1-1 and the human beta 3-subunit cDNA, we identified, respectively, a 6-kb and 3.8-kb transcript in the parathyroid (Fig. 6). The sizes of these transcripts were comparable to those of the rabbit skeletal muscle alpha 1-subunit and human beta 3-subunit transcripts, respectively (12, 14, 34).


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Fig. 6.   Northern blot analysis of the bovine parathyroid poly A+ RNA with cDNA probes prepared from a PTalpha 1-1 or a human beta 3-subunit cDNA probe as described in MATERIALS AND METHODS.

To assess the protein expression of alpha 1-subunits in parathyroid cells, we performed immunocytochemistry using an anti-pan-alpha 1 antiserum raised against an epitope that is conserved among alpha 1C-, alpha 1S-, and alpha 1D-subunits. Brown DAB staining for the alpha 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-alpha 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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Fig. 7.   Immunocytochemistry of bovine parathyroid glands. Tissue sections were stained with diaminobenzene (DAB) after incubation with anti-pan-alpha 1-subunit antibodies (a, b) or the same antibodies preabsorbed with peptide (c). Asterisks, arrowheads, and arrows depict parathyroid cells, arterioles, and venules, respectively.

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 (-)202-791 and R-BAY K 8644. As shown, adding (-)202-791 (10-6 M) had no significant effect on the sustained (>5 min) increase in [Ca2+]i induced by raising [Ca2+]o from 0.5 to 2.0 mM (Fig. 8A) or to 5.0 mM (data not shown). Increases in [Ca2+]i were, however, blocked by either La3+ (10-3 M) (Fig. 8A) or Gd3+ (data not shown). Pretreatment of cells with (-)202-791 (10-6 M) for 30-60 min also did not cause significant effects on [Ca2+]i (Fig. 8B). The increase in [Ca2+]i induced by high [Ca2+]o was blocked by the cation channel blocker Gd3+ (10-3 M) (Fig. 8B). Similar results were obtained using another L-type channel antagonist, R-BAY K 8644 (data not shown). These observations indicate that acute increases in [Ca2+]i in parathyroid cells due to high [Ca2+]o are not mediated by the activation of the dihydropyridine-sensitive channels. Instead, other La3+- or Gd3+-sensitive channels may be involved in mediating Ca2+ entry in these cells.


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Fig. 8.   Intracellular Ca2+ responses to changes in extracellular Ca2+ concentration ([Ca2+]o) and dihydropyridines in parathyroid cells. Intracellular [Ca2+] ([Ca2+]i) was determined as described in MATERIALS AND METHODS. Data are presented as values of the ratio of fluorescence at excitation of 340 and 380 nm (R340/380). A: changes of R340/380 in response to addition of (-)202-791 (10-6 M) and LaCl3 (10-4 M) after raising [Ca2+]o from 0.5 to 2.0 mM (top bar). Tracing shows an ensemble recording of changes in [Ca2+]i from a population of 24 cells. B: intracellular Ca2+ responses to raising [Ca2+]o from 0.5 to 2.0 mM (top bar) and addition of GdCl3 (10-4 M) in cells preincubated with (-)202-791 (10-6 M) for 30 min; (-)202-791 (10-6 M) was present throughout the recording (top bar). Tracing represents ensemble recordings from 34 cells. In both experiments, ionomycin (300 µM) and EGTA (20 mM) were added to obtain maximal and minimal R340/380, respectively. These data are representative of 5-6 coverslips of cells taken from 3 other cell preparations.

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 (10-9 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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Table 1.   Effects of different [Ca2+]0 and dihydropyridines on PTH release


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (approx 150 kDa) comparable to the skeletal alpha 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 alpha 1- and beta -subunits were expressed in these cells. Finally, cloning and RT-PCR data confirmed the presence of an alternatively spliced skeletal alpha 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 alpha 1-subunits may be developmentally regulated in certain tissues. For instance, alpha 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 alpha 1-subunit cDNA we cloned from the parathyroid appears to be an alternatively spliced product of the skeletal muscle alpha 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 alpha 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 alpha 1-subunit cDNA we cloned represents only approx 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 alpha 1-subunit cDNA. Alternatively, the protein product of the full alpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 281(1):E180-E189
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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