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
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ABSTRACT |
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
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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 M
. 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(
-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
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
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
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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).
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RESULTS |
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)
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).
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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
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
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.
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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 1 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.
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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.
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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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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.
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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 (
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 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 |
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 
-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.
 |
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