Parathyroid Ca2+-conducting currents are modulated by muscarinic receptor agonists and antagonists

Wenhan Chang1, Tsui-Hua Chen1, Stacy A. Pratt1, Benedict Yen2, Michael Fu3, and Dolores Shoback1

1 Endocrine Research Unit, Department of Medicine and 2 Department of Pathology, Veterans Affairs Medical Center, University of California, San Francisco, California 94121; and 3 Wallenberg Laboratory, Goteborgs University, Gothenburg, Sweden S-41345

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Parathyroid cells express Ca2+-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration approx 10-8 M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution. (+)-Muscarine enhanced the adenosine 3',5'-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed >90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.

calcium currents; calcium channel; calcium receptor; adenylate cyclase; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

IONIZED CALCIUM CONCENTRATION ([Ca2+]) directly regulates parathyroid hormone (PTH) secretion. High extracellular Ca2+ concentrations ([Ca2+]o) suppress both hormone release and biosynthesis, whereas low [Ca2+]o stimulates these processes (6, 31). [Ca2+]o is thought to modulate secretion by interacting with membrane Ca2+-sensing receptors (CaRs), which couple to the stimulation of phospholipase C activity (10) and inhibition of adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation (27). On the basis of pharmacological studies with ionophores and other agents that raise intracellular Ca2+ concentration ([Ca2+]i), it has been suggested that increments in this mediator are causally linked to the inhibition of PTH secretion (13, 25, 30). High [Ca2+]o-induced increases in inositol 1,4,5-trisphosphate (IP3) are temporally linked to the initial rapid phase of Ca2+ mobilization from intracellular stores (27). Ca2+ influx across the membrane is likely to be responsible for more long-term changes in [Ca2+]i that occur when cells are exposed to high [Ca2+]o (5). Studies from other laboratories suggest a role for L-type Ca2+ channels in regulating PTH release (25).

We previously characterized dihydropyridine-sensitive, cation-selective currents in parathyroid cells in which conductance was increased by raising [Ca2+]o (11), potentially through activation of the CaR. Although these currents can conduct Ca2+, they are not voltage gated like many dihydropyridine-sensitive L-type Ca2+ currents in excitable cells. In several systems, activation of muscarinic receptors (3, 24) regulates opening of Ca2+ channels that are dihydropyridine sensitive. Because previous studies indicated that muscarinic agonists could modulate PTH secretion (32, 38), we examined the effects of muscarinic receptor activation on the ionic currents in this system.

At least five subtypes of muscarinic receptors (M1-M5) have been identified in excitable and nonexcitable tissues (12, 18). Activation of M1, M3, and M5 receptors typically increases IP3 and diacylglycerol levels (4, 22). Activation of M2 and M4 receptors can either raise (19) or lower (34) cAMP levels, depending on the system. M2- and M4-receptor activation also modulates ion channel activity (18). In cardiac cells, activation of M2 receptors opens K+ channels and blocks Ca2+ channels (16). In neuronal cells, M4 receptors couple to blockade dihydropyridine-sensitive Ca2+ channels (3). As a subgroup of the G protein-coupled receptor superfamily, muscarinic receptors are linked to a striking variety of effector systems, depending on the cell type.

In parathyroid cells, we found that the agonists ACh and muscarine blocked the dihydropyridine-sensitive cation currents activated by raising [Ca2+]o (11). Muscarine enhanced the accumulation of the second messenger cAMP but not IP3. The ability of ACh to dampen this cation conductance depended on the presence of ATP in the patch pipette, suggesting a role for cAMP-dependent protein phosphorylation in regulating the function of the channels that conduct these Ca2+ currents. These results are compatible with the possibility that phosphorylation of either the channel, the CaR, or a regulatory molecule is required for muscarinic receptor-induced current suppression.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. ACh, atropine, and (+)-muscarine were purchased from Research Biochemicals International (Natick, MA). Media were prepared by the Cell Culture Facility of the University of California, San Francisco, CA. All channel blockers, salts, and chemicals were purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified.

Preparation of parathyroid cells. Isolated bovine parathyroid cells were prepared by collagenase and deoxyribonuclease digestion of parathyroid gland fragments (8) for patch clamping as previously described (11). Cell suspensions were incubated with the reagents specified for the determination of PTH release and inositol phosphate (IP) and cAMP accumulation (28). For electrophysiological studies, isolated cells were plated on no. 1 round cover glasses and incubated for 30 min at 37°C before recordings (11).

Whole cell recordings. Recording electrodes were prepared as previously described (11). Whole cell voltage clamping was performed, using glass pipettes with an electrical resistance of 1-4 MOmega . 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 acquired by using the following voltage-clamping protocol. Cells were held at -60 mV, and then a series of 150-ms test voltage pulses was applied at 2-s intervals from -100 to 120 mV in increments of 20 mV. The current traces presented in Figs. 1-6 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. The arrows represent zero current level. The Im values used for making Im-Vm plots are the arithmetic means of the currents recorded during the voltage pulses. Representative experiments are shown (see Figs. 1-6), and all experiments were performed on at least three cells at room temperature unless otherwise specified.

Electrode solutions. Most recordings were performed with a whole cell electrode solution (WCES) containing (in mM) 140 cesium 2-(N-morpholino)ethanesulfonic acid, 5 MgCl2, 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.4), 4 MgATP, 0.3 GTP, and a nucleotide-regenerating system (NRS; 14 mM phosphocreatine and 50 U/ml creatine phosphokinase) (2, 11). To test the role of intracellular ATP in channel regulation, WCES was replaced by perfusing the micropipette with an electrode solution that was the same as WCES except that the NRS and ATP were excluded (non-NR-WCES).

Micropipette perfusion. WCES, which typically bathes the interior of the cell, was replaced with the non-NR-WCES in several experiments to test the role of specific electrode solution constituents on the membrane conductances. The micropipette perfusion techniques we used were modified from published sources (33). Briefly, after the membrane-pipette seal and the whole cell recording configuration were established, we gently detached the cell from the coverslip and raised the cell above the bottom of the recording chamber by raising the micropipette holder. This provided the spatial freedom (0.2 cm in the vertical direction) for manually uncapping the tubing from the suction port of the micropipette holder. To deliver the new electrode solution, we inserted the slender drawn tip (<50 µm in diameter) of a 1-ml plastic syringe into the glass micropipette. The original electrode solution was replaced by perfusing 1 ml of the new electrode solution into the micropipette, using the syringe. To assure that the solution replacement was complete, the tip of the perfusing syringe was placed <1.5 mm from the tip of the recording pipette, and the volume of new electrode solution perfused was >10 times the volume of the recording pipette. Complete replacement of the electrode solution took 2-4 min. The electrode solution could be successfully replaced two or three times in most experiments. Recording was started 10 min after the electrode solution was replaced. This method did not disturb the pipette-membrane seal, because there was no change in seal resistance. We also checked that seal resistance was unchanged at the end of each experiment after adding the channel blockers Gd3+ or La3+.

Bath solutions and extracellular bath perfusion. All bath solutions (BS) contained 10 mM HEPES (pH 7.4) and 10 mM tetraethylammonium ion (TEA+) to block endogenous K+ currents (2). Various [Ca2+] in the BS (0.7-90 mM) were achieved by the addition of Ca acetate. Acetate was the anion charge carrier so as to minimize recordings from endogenous Cl- currents (11). Osmolarity of the BS was adjusted to approx 330 mosmol/l with sucrose as needed. Each BS is specified by the concentration of its major cation species as follows: 90 Ca/10 TEA acetate BS contains (in mM) 90 Ca2+, 10 TEA+, 190 acetate-, and 10 HEPES (pH 7.4); and 0.7 Ca/10 TEA acetate BS contains (in mM) 0.7 Ca2+, 10 TEA+, 11.4 acetate-, 10 HEPES (pH 7.4), and 267 sucrose.

Recordings were made in a Lucite perfusion chamber with a volume of 0.8 ml. To replace the BS, the recording chamber was perfused with 15 ml of each new solution at a rate of approx 10 ml/min. Receptor analogs and channel blockers were added by mixing a concentrated stock solution to the appropriate BS, which was then delivered to the recording chamber. All recordings were initiated at least 10 min after delivery of a given BS.

Reverse transcriptase-polymerase chain reaction. Three hundred to four hundred base pair cDNA fragments encoding portions of the third intracellular loop of M1-M5 receptors were amplified with standard reverse transcriptase-polymerase chain reaction (RT-PCR) techniques (20). The primers used for M2, M3, and M4 receptors (Table 1) were derived from the sequences of the respective receptor cDNAs cloned from bovine adrenal gland and brain cDNA libraries (23) (GenBank accession numbers: M2-L27102, M3-U08286, M4-L27104). Because there were no available sequences for bovine M1- and M5-receptor cDNAs, we designed primers based on conserved sequences in these receptors among different species (GenBank accession numbers: human M1-X15263, rat M1-M16406, mouse M1-J04192, and porcine M1-X04413; and human M5-M80333 and rat M5-M22926) (see Table 1).

                              
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Table 1.   Nucleotide sequences of primers used to amplify bovine parathyroid cDNA from IC3 of muscarinic receptor subtyes

PCR was performed on the first-strand cDNA reverse transcribed from ~0.5 µg bovine parathyroid poly (A)+ RNA in a 100-µl reaction containing MgCl2 (1.5 mM), 2'-deoxynucleoside 5'-triphosphate set (0.3 mM), and primers (1-2 µg). The reaction was initiated by adding 2.5 U Taq DNA polymerase (GIBCO BRL, Gaithersburg, MD) at 95°C in a Twin Block System thermal cycler (Ericomp, San Diego, CA). The following conditions were used: 95°C for 1 min, 55 or 58°C for 1 min, and 72°C for 3 min for 30-40 cycles. The reaction ended with a 7-min extension at 72°C. PCR products were electrophoresed on agarose gels, visualized by ethidium bromide staining, and purified with the use of Qiaex DNA extraction kits (Qiagen, Chatsworth, CA). PCR fragments were blunt ended and subcloned into the Sma I site of plasmid Bluescript II SK(-) (Stratagene, La Jolla, CA) and sequenced with the use of Sequenase II (US Biochemical, Cleveland, OH).

Western blotting. Membrane proteins were prepared from newborn calf parathyroid glands and heart as described (15). Parathyroid and heart membrane proteins (150 µg) were electrophoresed on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (21). Membranes were blocked with a solution (Blotto) containing tris(hydroxymethyl)aminomethane buffer (10 mM, pH 8.0), skim milk (5%, wt/vol), NaCl (150 mM), and Tween 20 (0.05%, wt/vol) at room temperature for 1-2 h. Membranes were incubated with either affinity-purified anti-M2-receptor antibodies (anti-M2-EC2; 10 nM), anti-M2-EC2 preincubated with 300 nM M2-EC2 peptide, or nonimmune rabbit immunoglobulin G (IgG) in Blotto solution without Tween 20 overnight at 4°C. The anti-M2-EC2 receptor antiserum was raised against a synthetic peptide corresponding to part of the second extracellular loop of the human M2 receptor [M2-EC2 peptide, VRTVEDGECYIQFFSNAAVTFGTAI (15)]. After washing the membranes three times with Blotto, we incubated them with peroxidase-conjugated goat anti-rabbit IgG (1:5,000; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Standard enhanced chemiluminescence assay kits were used for signal detection (Amersham Life Science, Arlington Heights, IL).

Immunocytochemistry. Newborn calf parathyroid glands were obtained and frozen in liquid nitrogen. Frozen sections were cut to a thickness of 4 µm. After being mounted on slides, sections were treated with 0.6% H2O2 in 80% methanol to reduce endogenous peroxidase activity and blocked with a solution of 3% albumin and 10% goat serum in phosphate-buffered saline (PBS). Sections were then incubated with 100-200 µl of either anti-M2-EC2 (10 nM), anti-M2-EC2 + M2-EC2 peptide (300 nM), or nonimmune rabbit IgG (100 nM) in PBS overnight at 4°C. After being rinsed in PBS, sections were treated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:50) at room temperature for 30 min. Tissue sections were washed and subjected to diaminobenzidine (DAB) staining, using SigmaFast DAB tablets (Sigma).

Measurement of PTH release, IPs, and cAMP. Total IP3, inositol 1,4-bisphosphate, and inositol 1-phosphate1 were separated from extracts of parathyroid cells after labeling of membrane polyphosphoinositides with myo-[3H]inositol as previously described (29). Changes in cAMP accumulation were assessed in parathyroid cells incubated with forskolin to amplify basal cAMP content in the presence or absence of (+)-muscarine. cAMP was determined by radioimmunoassay of cellular extracts from these experiments (28). PTH release was measured from cells treated with or without muscarine for 30 or 60 min at 37°C (28). Statistical significance was determined by paired or unpaired t-test and analysis of variance, using StatView II computer software (Abacus Concepts, Berkeley, CA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Muscarinic receptor agonists and antagonists modulate Ca2+-conducting currents. We previously characterized two types of Ca2+-conducting currents in bovine parathyroid cells distinguished by their sensitivity to channel blockers (11). Type 1 Ca2+ currents, constituting at least 50% of the whole cell Gm, increased with raising [Ca2+]o and were blocked by the dihydropyridine nifedipine [50% inhibitory concentration (IC50) approx 3 × 10-8 M] (Fig. 1). The residual Ca2+ currents (type 2) were suppressed by the inorganic blocker Gd3+ or La3+ (Fig. 1). Both currents were cation nonselective and not classically voltage gated (11). These pharmacological and biophysical characteristics were evident in all recordings with gigaohm membrane-pipette seals.


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Fig. 1.   Pharmacological characterization of Ca2+-conducting currents in bovine parathyroid cells. A: whole cell patch-clamp recordings were made as described in MATERIALS AND METHODS from a cell perfused with 0.7 Ca/10 TEA or 90 Ca/10 TEA acetate bath solution (BS; see Bath solutions and extracellular bath perfusion for composition) in absence (control) or presence of channel blocker nifedipine (Nif; 3 × 10-4 M) or Gd3+ (3 × 10-3 M). Arrows, 0 current level. B: membrane conductance (Gm) derived from slopes of membrane current (Im)-membrane potential (Vm) plots shown in A. Pipette solution: whole cell electrode solution (WCES).

Because muscarinic receptor activation modulates cation-selective (14) and dihydropyridine-sensitive Ca2+ channels (3, 24) in other cells and because parathyroid cells respond to muscarinic agonists (32, 38), we tested the effects of receptor agonists and antagonists on Ca2+-conducting currents in this system. As shown in Fig. 2, ACh suppressed Im and Gm in a dose-dependent manner with an IC50 of approx 10-8 M. This result was confirmed in another experiment in a different cell preparation (data not shown). The reversal potential derived from Im-Vm plots did not shift significantly with the application of ACh (data not shown), suggesting that ACh was not affecting the ion selectivity of these currents. The effect of ACh was apparent within 10 min of its addition to the bath and was reversed by its removal from the bath (Fig. 2A). There was complete inhibition of type 1 currents by maximal doses of ACh (1 µM) in approx 70% of the cells studied. The degree of blockade was equivalent with ACh and nifedipine (Fig. 2A, traces iv and vi), and their effects were not additive (data not shown). ACh profoundly blocked the ability of raising [Ca2+]o from 0.7 to 90 mM to enhance Im and Gm (Fig. 3), and the effects were reversible (n = 4 cells). These findings support the idea that ACh suppressed the type 1 Ca2+ conductance we previously identified in these cells (11).


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Fig. 2.   ACh suppressed Nif-sensitive Ca2+ currents in a dose-dependent manner. A: whole cell recordings from a cell perfused with 90 Ca/10 TEA acetate BS containing various concentrations of ACh (10-9 to 10-7 M, traces i-iv). Effect of ACh is reversible (trace v) and comparable in magnitude to that of Nif (10-5 M, trace vi). Arrows, 0 current level. B: Gm in response to different doses of ACh. Pipette solution: WCES.


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Fig. 3.   ACh blocked the ability of raising extracellular Ca2+ concentration ([Ca2+]o) to enhance Im and Gm. A: whole cell recordings from a cell perfused with BS containing various concentrations of Ca acetate (0.7-45 mM) in presence or absence of ACh (10-5 M). Arrows, 0 current level. B: Gm in response to raising [Ca2+]o in presence or absence of ACh. Pipette solution: WCES.

To confirm that the effects of ACh were due to an interaction with muscarinic cholinergic receptors, we tested another pharmacological agonist and an antagonist. The agonist (+)-muscarine suppressed the baseline Im at 0.7 mM Ca2+ by >30% in each experiment as well as the Im induced by raising [Ca2+]o up to 90 mM (Fig. 4). (+)-Muscarine was less potent in suppressing the activation of Im at >= 22.5 mM, which was also confirmed in two additional experiments (data not shown). The muscarinic receptor antagonist atropine blocked the inhibitory effects of ACh on type Im by >70% (n = 2 cells; Fig. 5). When atropine was removed from the perfusate for ~15 min, the ability of ACh to suppress Im was restored, indicating that the response to ACh was intact (Fig. 5D), as was the inhibitory effect of the nonselective cation channel blocker La3+ (3 × 10-3 M; data not shown).


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Fig. 4.   (+)-Muscarine mimicked ability of ACh to suppress high [Ca2+]o-induced Ca2+-conducting currents. A: whole cell recordings from a cell superfused with BS containing various concentrations of Ca acetate (0.7-45 mM) in presence or absence of (+)-muscarine (10-5 M). Arrows, 0 current level. B: Gm in response to increasing [Ca2+]o in presence or absence of (+)-muscarine. Pipette solution: WCES.


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Fig. 5.   Atropine blocked inhibitory effect of ACh on Ca2+-conducting currents. Whole cell recordings were performed sequentially in control BS (90 Ca/10 TEA acetate BS) containing no ACh analogs (A), atropine (10-5 M; B), atropine (10-5 M) + ACh (5 × 10-6 M) (C), and ACh (5 × 10-6 M; D). Each recording was made at least 10 min after each BS was delivered. Pipette solution: WCES. Arrows, 0 current level.

Muscarinic receptor activation in other cells is known to influence cAMP, [Ca2+]i, and diacylglycerol (18, 22, 36), second messengers that enhance the activities of several classes of protein kinases. To determine whether ACh-induced suppression of Ca2+-conducting currents depended on protein phosphorylation, we removed ATP from the electrode solution by using the non-NR-WCES. When the pipette solution was WCES, which contains ATP, perfusion of the bath surrounding the cell with ACh suppressed Im and Gm (Fig. 6A, traces i and ii). When we perfused the pipette with non-NR-WCES, which lacks ATP and the ability to generate it, and ACh was still present in the bath, the ability of ACh to inhibit Im was reversed (Fig. 6, A, trace iii, and B). When only ATP was added back to the non-NR-WCES in the pipette, the ability of ACh to reduce Im and Gm was again demonstrated (Fig. 6, A, trace iv, and B). Thus the blockade of currents by ACh depended on the presence of ATP in the pipette solution. The removal of ATP from the pipette solution also interfered with the ability of ACh to suppress high [Ca2+]o-induced increments in Gm (Fig. 6B, traces i and iii). Comparable results were observed in three other cells. These findings indicate a key role of ATP and likely protein phosphorylation in mediating the effects of ACh on these currents and their response to changes in [Ca2+]o.


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Fig. 6.   Removal of ATP from electrode solution reversed inhibitory effect of ACh on Im and Gm. A: whole cell recordings were performed in 90 Ca/10 TEA acetate BS without (i) or with (ii, iii, and iv) ACh in bath. Patch-pipette perfusion solutions were WCES (i and ii), non-NR-WCES (WCES without nucleotide-regenerating system or ATP; iii), or non-NR-WCES + 4 mM MgATP (iv). Each recording was taken at least 10 min after new BS or electrode solution was delivered. Arrows, 0 current level. B: Gm in response to increments in [Ca2+]o in absence (i) or presence (ii, iii, and iv) of ACh, with different pipette solutions described for A.

Effects of muscarinic agonists on cAMP, IPs, and PTH release. To determine whether second messengers might be involved in the mechanism by which ACh blocks Ca2+-conducting currents in parathyroid cells, we measured cAMP and IP levels. The latter serves as an index of polyphosphoinositide turnover. Incubation of these cells with (+)-muscarine (10-5 M) in medium containing 1 mM Ca2+ for 10 min increased cAMP accumulation by 29.0 ± 7.3% (n = 27, 3 cell preparations), whereas 30 mM NaF suppressed cAMP accumulation by 66.2 ± 5.5% (n = 9; Fig. 7A). We also verified the inhibitory effects of raising [Ca2+]o on cAMP production in each cell preparation to confirm the integrity of cAMP responses in these cells. Raising [Ca2+]o from 0.5 to 10.0 mM suppressed cAMP production in these cells by 39.3 ± 7.1% (n = 18, Fig. 7B). Exposing parathyroid cells to ACh (10-5 M) for up to 20 min, however, did not change total IP levels (Table 2). Furthermore, ACh had no effect on the IP responses to raising [Ca2+]o from 0.5 to 3.0 mM (data not shown).


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Fig. 7.   (+)-Muscarine enhanced cAMP production in parathyroid cells. Cells were incubated in medium containing 3 µM forskolin for 10 min before treatments. A: effects of 10 µM (+)-muscarine or 30 mM NaF on cAMP production at [Ca2+]o = 1 mM. B: effects of raising [Ca2+]o from 0.5 (solid bar) to 10 mM (open bar) on cAMP production. * P < 0.01; ** P < 0.005.

                              
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Table 2.   Effects of ACh on total IP production in parathyroid cells

PTH release can be altered by changes in cAMP and Ca2+ mobilization (6). PTH release from cells treated with muscarine (10 µM) for 30 min declined by 15-20% (n = 2). The effect, however, was not sustained at the 60-min time point (data not shown).

PCR amplification of muscarinic receptor cDNAs in the parathyroid. The electrophysiological studies described above suggested that muscarinic receptors were expressed in the parathyroid. To determine which receptor subtype(s) is present, we performed RT-PCR with five sets of primers designed to amplify sequences in the third intracellular loop of the muscarinic receptors (M1-M5). Primers homologous to M2- and M4-receptor sequences successfully amplified cDNA fragments by RT-PCR, using bovine parathyroid cDNAs as the template DNA. The nucleotide and derived peptide sequences of these PCR fragments showed 91 and 93% identity, respectively, with human M2 receptors (Fig. 8A). PCR fragments encoding the third cytoplasmic loop of a putative M4 receptor demonstrated 85 and 88% identity in the nucleotide and derived peptide sequences, respectively, compared with human M4 receptors (Fig. 8B). Primers designed to amplify M1-, M3-, and M5-receptor cDNAs did not generate cDNAs of the expected sizes. We sequenced other PCR fragments generated in these reactions to determine whether they were the result of false priming or the existence of alternatively spliced forms of these receptors. The cDNAs amplified with M1-, M3-, and M5-receptor primer pairs showed no homology to any known muscarinic receptors, indicating they were likely due to false priming (data not shown).


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Fig. 8.   Third intracellular loop of M2 receptor. A: alignments of nucleic acid and derived amino acid sequences of amplified bovine parathyroid (PT) M2-receptor cDNA with human M2-receptor sequences. B: alignments of PT M4-receptor cDNA with human M4-receptor sequences. Primer sequences used in cDNA amplifications are underlined. Numbers in parentheses are nucleotide sequence numbers.

Immunodetection of M2 muscarinic receptors. Using an antibody raised against the second extracellular loop of the human heart M2 receptor (anti-M2-EC2) (15), we demonstrated the expression of M2-receptor protein in the parathyroid. Immunoblots of membrane protein with anti-M2-EC2 demonstrated a protein band (approx 100 kDa) in bovine parathyroid and ventricle that was not present in samples incubated with nonimmune rabbit serum or with anti-M2-EC2 preincubated with M2-EC2 peptide (Fig. 9). The faint bands, appearing only in lanes containing parathyroid membrane protein after preabsorption of the antisera with peptide, were distinctly smaller than the M2-receptor bands present in ventricle and parathyroid gland and were thought to be nonspecific. These results indicate that proteins isolated both from parathyroid and ventricle react with antibodies raised against human cardiac M2 receptors.


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Fig. 9.   Immunoblots of whole cell lysates prepared from bovine (b) cardiac and parathyroid tissues. A band of approx 100 kDa was detected in lanes containing proteins from ventricle and parathyroid gland by anti-M2-receptor antibodies (anti-M2-EC2; left) but not by anti-M2-EC2 preincubated with M2-EC2 peptide (middle) or nonimmune rabbit immunoglobulin (Ig) G fraction (right).

M2-receptor protein was localized by indirect immunocytochemistry. Incubation of parathyroid gland sections with anti-M2-EC2 revealed a strong cell surface staining pattern (Fig. 10, A and B). The staining was specific, since it was blocked by preincubating the antibodies with M2-EC2 peptide (Fig. 10C) and was not present in sections exposed to nonimmune rabbit IgG (Fig. 10D).


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Fig. 10.   Immunocytochemistry of bovine parathyroid glands. Tissue sections were stained with diaminobenzidine after incubation with anti-M2-EC2 (A and B), anti-M2-EC2 + M2-EC2 peptide (C), or nonimmune rabbit IgG fraction (D). Bars = 30 µm (A) and 10 µm (B-D). Arrows, plasma membrane staining.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A variety of extracellular signals regulates PTH release, including ions like Ca2+ and Mg2+ and the neurotransmitters epinephrine, dopamine, and ACh (5, 6). Divalent cations are thought to modulate secretion by interacting with a recently identified Ca2+ receptor (7, 9). This receptor couples changes in the [Ca2+]o to second messenger production (7, 9) and ion channel activation (39) in transfected cells expressing receptor cDNA. In whole cell recordings of parathyroid cells, we previously characterized voltage-insensitive, cation-selective currents that are carried by Ca2+. Their amplitude increased with raising [Ca2+]o, including Ca2+ levels within the physiological range (11). Responsiveness of these currents to changes in [Ca2+]o depended on the presence of GTP in the patch pipette, suggesting the involvement of a G protein in coupling the membrane Ca2+-sensing mechanism to the channel proteins (11). Whether Ca2+ receptors couple directly to these channels through activated G-protein subunits or whether the channels are opened by second messengers generated by stimulating the Ca2+ receptor remains an important but unanswered question.

The regulation of PTH secretion by agents such as dopamine, epinephrine, and ACh is thought to occur through membrane receptors. The muscarinic cholinergic receptor family is particularly known for its ability to activate or inhibit multiple downstream effector pathways. These effectors include phospholipase C and adenylate cyclase as well as K+, nonselective cation, and L-type Ca2+ channels, depending on the cell system (18). Because previous studies demonstrated that muscarinic agonists could affect PTH secretion (32, 38) and that the gland was innervated by the autonomic nervous system (17), we tested the effects of muscarinic receptor agonists on Ca2+-conducting currents. Our studies showed that receptor activation was coupled to the inhibition of the dihydropyridine-sensitive cation-selective conductance and interfered with the ability of high [Ca2+]o to enhance this conductance. The receptor antagonist atropine blocked the effect of ACh. We further adapted a patch-pipette perfusion technique to these cells and demonstrated that the effects of ACh depended on the presence of ATP in the intracellular perfusion solution. Taken together, these findings suggest that muscarinic receptor activation couples to the inhibition of a cation current and that this process may involve protein phosphorylation.

We used a PCR-based approach to identify the muscarinic receptor subtype(s) in the parathyroid. Primers were designed to amplify the putative third intracellular loops of M1-M5 subtypes. In these receptors, this region of the molecule is important in mediating signal transduction (37). The primers chosen to amplify M2- and M4-receptor cDNAs were based on available partial sequences from bovine adrenal gland that were highly conserved compared with other cloned M2 and M4 receptors from human, rat, and porcine tissues. The primers used for M3-receptor cDNA amplification were derived from bovine adrenal gland and brain M3-receptor cDNA sequences. Because comparable cDNAs for M1 and M5 receptors were not available from bovine tissues, the primers we used were based on conserved sequences among human, rat, and porcine receptor cDNAs. We consistently amplified only M2- and M4-receptor sequences in our RT-PCR studies. Although the annealing temperature, MgCl2 concentration, and cycle number were varied extensively in an effort to amplify M1-, M3-, and M5-receptor cDNAs, PCR products with sequences homologous to known muscarinic receptor cDNAs were never obtained. Our ability to exclude expression of M1-, M3-, and M5-receptor cDNAs in the parathyroid is limited by scant information on tissue distribution and abundance of these receptors in the bovine species and the unavailability of positive control tissues, such as bovine brain, for the development and testing of alternate primer pairs. On the basis of these studies, we conclude that M2- and/or M4-receptor subtypes are the most likely candidates for mediating the effects of muscarinic agonists and antagonists on the Ca2+-conducting currents in the parathyroid.

Western blotting and immunocytochemistry with an anti-M2-EC2 peptide confirmed the presence of M2-receptor protein in the parathyroid. Anti-M2-receptor antiserum detected a protein band of ~100 kDa in membrane preparations from bovine parathyroid gland and ventricle. The size of this protein band compares favorably with sizes reported for the M2 receptor (~80 kDa) in rat ventricular membranes (15) and calf brain (1). Incubation of the same antiserum with tissue sections from the parathyroid gland revealed a distinct cell surface staining pattern in a distribution compatible with chief cell localization. These Western blot and immunocytochemistry results are compatible with the presence of an M2 receptor specifically in the parathyroid for the following reasons. 1) Cell surface staining and the presence of the protein band on immunoblots disappeared when antiserum was preabsorbed with M2-receptor peptide. 2) Staining of gland sections and the ~100-kDa protein were not present when nonimmune rabbit serum was substituted for anti-peptide antiserum. 3) Sequence comparisons of the second extracellular loops of the five muscarinic receptor subtypes indicate at most only 50% conservation of peptide sequence between M2 and M1, M3, M4, or M5 receptors from both human and rat tissues (15). This would render cross-reactivity of the antiserum we used with other muscarinic receptor subtypes unlikely. Because we were unable to obtain antisera directed against M4-receptor epitopes, comparable studies could not be performed to localize M4-receptor protein further in this tissue. These findings support the conclusion that M2 and possibly M4 receptors mediate the actions of muscarinic agonists and antagonists in the parathyroid.

The hypothesis that M2 and possibly M4 receptors may be responsible for suppressing dihydropyridine-sensitive cation currents in the parathyroid is compatible with the specific functional properties of these receptor subtypes in neurons (35) and in AT-20 pituitary cells (24). Muscarinic receptor subtypes other than M2 and M4, however, can couple to the regulation of L-type Ca2+-conducting or nonselective cation channels, depending on the specific cell under study (18). Clearly, the degree of diversity in functional responses resulting from muscarinic receptor activation makes it difficult to predict with certainty which receptor subtype(s) is expressed in a given tissue based on the currents affected by pharmacological agonists or antagonists.

Muscarinic receptors also activate phosphoinositide hydrolysis and modulate changes in adenylate cyclase activity (4, 19, 22, 34). Our observation that the suppression of Ca2+-conducting currents by ACh required the presence of ATP in the patch pipette suggested that protein phosphorylation(s) may be crucial for current blockade. Increases in cAMP accumulation by (+)-muscarine in parathyroid cells further implicated cAMP-dependent pathway(s) as being involved in coupling between muscarinic receptors and the currents. This notion was supported by our recent finding that intracellular perfusion of cAMP inhibits the Ca2+-conducting currents in a reversible dose- and ATP-dependent manner (unpublished data). These observations lead to the idea that activation of ACh receptors may suppress Ca2+-conducting currents through cAMP-dependent phosphorylation of ion channel proteins and/or their regulators.

In many systems, activation of M2 and M4 receptors lowers cAMP levels (4, 36, 37). High concentrations of muscarinic agonists, however, enhanced cAMP production in Chinese hamster ovary cells transfected with the M4-receptor cDNA (19), indicating that the regulation of cAMP production by muscarinic receptors may differ, depending on the concentration of agonists and/or cell types. Our observation that muscarine can increase cAMP in parathyroid cells is not the typical prediction, but it is supported by work in other systems. We also found that muscarine had transient inhibitory effects on PTH release, which was not predicted either, since increasing cAMP content typically stimulates PTH release (5, 6). These findings, however, are in agreement with the studies of Williams et al. (38), who examined PTH secretion from gland slices. Whether the effects of muscarine we observed on cAMP and PTH release occur in the in vivo setting remains to be further explored.

The observation that ACh blocked the ability of high [Ca2+]o to enhance the Ca2+-conducting currents was also unexpected. This finding suggested that there may be an interaction between muscarinic and Ca2+ receptors in these cells, leading ultimately to current blockade. At present, the molecular basis for this receptor interaction is unknown. Whether muscarinic receptor-induced production of second messengers or the release of specific beta gamma -subunits mediates the inhibitory effects of muscarinic receptor activation on [Ca2+]o-induced enhancement of this conductance awaits further studies in defined reconstitution systems. The present findings indicate that patch-pipette perfusion techniques adapted for these studies would provide a way to test these possibilities in the parathyroid cell system.

    ACKNOWLEDGEMENTS

We thank Drs. John Imboden and Theodora Mauro for helpful discussions and Dr. I-Hsiung Tang at the University of California, Davis, for technical advice.

    FOOTNOTES

D. Shoback is supported by a Department of Veterans Affairs Merit Review, the Northern California Arthritis Foundation, and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43400.

1 IP3, inositol 1,4-bisphosphate, and inositol 1-phosphate were not resolved further into isomeric forms in these studies.

Address for reprint requests: D. Shoback, 111N, Endocrine Research Unit, Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121.

Received 4 December 1996; accepted in final form 7 July 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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