Regulation of extracellular calcium-activated cation currents by cAMP in parathyroid cells

Wenhan Chang, Tsui-Hua Chen, Stacy Pratt, and Dolores Shoback

Endocrine Research Unit, Department of Veterans Affairs Medical Center, Department of Medicine, University of California, San Francisco, California 94121

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Parathyroid cells express Ca2+-sensing receptors that couple changes in the extracellular Ca2+ concentration ([Ca2+]o) to increases in the intracellular free Ca2+ concentration ([Ca2+]i) and to the suppression of parathyroid hormone secretion. Using whole cell patch clamping, we previously identified voltage-independent Ca2+-conducting currents in bovine parathyroid cells that increased with rising [Ca2+]o and were blocked by Cd2+ and nifedipine. Because cAMP-dependent phosphorylation regulates dihydropyridine-sensitive Ca2+ channels in other systems, we tested whether cAMP modulates these currents. At 0.7 mM Ca2+, nonselective Ca2+-conducting currents were suppressed by 30-50% when the recording pipette was perfused with cAMP. High-[Ca2+]o-induced increases in membrane currents were also abrogated. The effects of cAMP were reversible and dose dependent (3 × 10-9 to 3 × 10-3 M) and required ATP in the pipette solution. Perfusion of the cell interior with the catalytic subunit of protein kinase A mimicked the effects of cAMP, as did perfusion of the bath with the adenylate cyclase activator forskolin. These findings support the idea that cAMP-dependent phosphorylation suppresses high-[Ca2+]o-induced cation currents and may play a role in regulating ion fluxes in parathyroid cells.

parathyroid hormone secretion; nonselective cation currents; calcium receptor; dihydropyridine; calcium channel; calcium influx

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EXTRACELLULAR Ca2+ concentration ([Ca2+]o) determines the rate of parathyroid hormone (PTH) secretion in vivo. Low [Ca2+]o maximally stimulates secretion, whereas high [Ca2+]o suppresses PTH release (2, 25). Parathyroid cells respond to changes in [Ca2+]o by altering the levels of critical second messengers, including cAMP, inositol trisphosphate, diacylglycerol, and intracellular Ca2+ concentration ([Ca2+]i) (2, 19, 25, 29). The accumulation of inositol 1,4,5-trisphosphate induced by raising [Ca2+]o is likely to play a key role in the initial mobilization of Ca2+ from intracellular stores (1). Sustained Ca2+ mobilization, however, requires membrane Ca2+ influx, presumably through the opening of ion channels. The nature of the channels responsible for the Ca2+ influx due to the raising of [Ca2+]o in the parathyroid is controversial, and their molecular identity is unknown.

Certain pharmacological studies have implicated dihydropyridine-sensitive ion channels in the regulation of PTH secretion (11, 26). Other studies, using antisera directed against alpha 1-subunits of L-type Ca2+ channels, support a role for L-type channels in modulating Ca2+ influx and PTH release (12). Using whole cell patch clamping, we recorded dihydropyridine-sensitive, nonselective cation currents that conduct Ca2+ and are activated by raising [Ca2+]o (6). This membrane conductance may be a component of the mechanism responsible for Ca2+ influx in this system.

The activity of the Ca2+ channel is regulated by protein kinases. cAMP-dependent phosphorylations, initiated by protein kinase A (PKA), are one pathway implicated in the control of ion channels, including the dihydropyridine-sensitive Ca2+ channels of many excitable cells (15-17, 31). In the present study, we found that internal perfusion of parathyroid cells with cAMP or the catalytic subunit of PKA (PKA-CS) and perfusion of the bath with forskolin blocked Ca2+-conducting currents. These effects required the presence of ATP in the pipette, suggesting that blockade of these currents involves phosphorylation of either the channel protein and/or a critical regulator. These findings raise the possibility that ion influx in the parathyroid via these channels is controlled by cAMP-induced phosphorylation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

Chemicals in the electrode and bath solutions, channel blockers, the sodium salts of cGMP and cAMP, forskolin, and PKA-CS were purchased from Sigma (St. Louis, MO). The acetoxymethyl ester of fura 2 was from Molecular Probes (Eugene, OR). Culture media were prepared by the Cell Culture Facility of the University of California, San Francisco. Recording micropipettes were pulled from thin-walled borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) by an electrode puller (model P-87, Sutter Instrument, Novato, CA) and polished using a Narishige MF-9 microforge (Technical Instruments, San Francisco, CA) as previously described (6, 7).

Cell Preparation

Dispersed bovine parathyroid cells were prepared (4) for patch clamping as described (6, 7). After the cell pellet was washed three times with Ca-, Mg-, and NaHCO3-free Eagle's medium containing HEPES (20 mM, pH 7.4) (cell medium) supplemented with CaCl2 (1 mM) and MgSO4 (1 mM), the cells were plated on no. 1 round microscope coverglasses (Fisher Scientific, Santa Clara, CA) and incubated for 30 min at 37°C before recording. For the measurement of [Ca2+]i, cells were equilibrated in the cell medium noted above supplemented with 0.5 mM CaCl2, 0.5 mM MgSO4, and 0.2% BSA (wt /vol) .

Solution Composition

Pipette solutions. Recording pipettes in most experiments were first filled with a whole cell electrode solution (WCES) containing (in mM) 140 cesium methanesulfonate, 5 MgCl2, 10 EGTA, 10 HEPES (pH 7.4), 4 MgATP, 0.3 GTP, and a nucleotide-regenerating system [NRS; 14 units/ml phosphocreatine and 50 units/ml creatine phosphokinase (6, 7)]. In this solution, Cs+ was the major charge carrier for outward currents. In certain experiments, we excluded both the NRS and ATP (NRS-free WCES) to examine the effects of these components on membrane currents.

Bath solutions. In the bath solution (BS), acetate was the major anion present to minimize the recording of Cl- currents. Ca2+ was the major carrier of the inward currents. Tetraethylammonium ion (TEA+; 10 mM) was included in these solutions to block K+-selective currents and also to act as a charge carrier (6, 7). All solutions were buffered with HEPES (10 mM, pH 7.4), and osmolarity was adjusted to approx 330 mosM with sucrose as needed. Each BS is designated by the major cations that it contains. Their compositions are specified in Table 1.

                              
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Table 1.   Ion compositions of bath solutions

Whole cell recordings were made in Lucite perfusion chambers with a volume of 0.8 ml. Substitution of the BS was made by perfusion of the recording chamber with 15 ml of the desired solution at a rate of approx 10 ml/min. Channel blockers were delivered to the perfusion chamber by mixing a stock solution (10-1 M) with the appropriate BS. All recordings were initiated at least 5 min after delivery of a given BS.

Patch-Clamping Protocols and Data Analysis

Whole cell patch clamping was conducted as described (6, 7). Briefly, thin-walled borosilicate glass pipettes with an electrical resistance of approx 1 MOmega were used. We established the whole cell recording configuration by applying short suction pulses to the patch pipette after achieving the cell-attached configuration to rupture the membrane. The attainment of the whole cell configuration was confirmed by either a sudden increase in membrane capacitance and/or an increase in membrane currents. To avoid analyzing nonspecific leakage currents due to damage of the pipette-membrane seal by suction pulses or membrane rupture, we examined the electrical seals of the membrane patches at the end of each experiment by measuring the series resistance and the ability of La3+ or Gd3+ to block membrane conductance (6, 7). Only recordings of cells that maintained seal resistances of >1 GOmega were included for analysis. All recordings were conducted at room temperature and were begun at least 10 min after the whole cell recording configuration was established.

Membrane potential (Vm) was controlled, and membrane current (Im) values were detected by an Axopatch amplifier (Axon Instruments, Foster City, CA) as described (6). Data were digitized (100 kHz), recorded onto a microcomputer, and analyzed with the use of pCLAMP and AxoGraph software.

The voltage-clamping protocol for acquiring the membrane current-voltage relationship (Im-Vm) plots is shown in Fig. 1A and was as follows. Each run of voltage clamping was initiated with the resting membrane potential held at -60 mV. Test voltage pulses of 150-ms duration were applied to the recording pipette every 2 s in increments of 20 mV (from -100 to +120 mV). The presented current traces were recorded from 20 ms before to 25 ms after each applied voltage pulse. Inward (+) currents, from the bath to the cytoplasmic compartment of the cell, are presented as downward deflections in the current traces. Current traces represent the arithmetic means of the currents recorded during three consecutive runs of the same voltage protocol. Im was calculated as the average current during a given test voltage pulse for determining the Im-Vm. The membrane conductance, designated as Cm, was calculated from the slope of the linear regression of the Im-Vm. Each experiment was performed on at least three cells from different preparations.


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Fig. 1.   Suppression of both inward and outward currents in parathyroid cells by intracellular perfusion of cAMP. Details of voltage-clamping protocol for whole cell recordings (A), data analysis, and presentation are described in MATERIALS AND METHODS. B: cell was bathed in 90 mM Ca-10 mM tetraethylammonium (TEA) acetate bath solution (BS). The recording pipette was filled with whole cell electrode solution (WCES), and whole cell currents were recorded. C: electrode solution (ES) was then replaced with a WCES with added cAMP (3 × 10-4 M; WCES+cAMP) with use of micropipette perfusion techniques described in MATERIALS AND METHODS. D: after recordings were obtained, WCES+cAMP was replaced with WCES [WCES (rinse)]. E: to ensure that electrical seal was not damaged by pipette perfusion, Gd3+ (3 × 10-3 M) was added to BS to block membrane conductance and evaluate integrity of electrical seal. This experiment is representative of similar studies in 8 cells. Signals were low-pass filtered with cutoff frequency (fc) = 10 kHz. B-E: arrows indicate zero current level. Vtest, test voltage pulses; Vh, resting membrane potential.

Micropipette Perfusion

Micropipette perfusion techniques adapted for parathyroid cells were described previously (7) and are illustrated in Fig. 2. After the whole cell configuration was established, the cell was gently detached from the coverslip and lifted up by raising the micropipette holder (World Precision Instruments) with a micromanipulator (Technical Instruments, San Francisco, CA) (Fig. 2). This provided the spatial freedom (0.2 cm in vertical direction) for manually uncapping the tubing from the suction port of the micropipette holder (Fig. 2). The slender drawn tip (<50 µm in diameter) of a 1-ml plastic syringe was placed inside the hub of the micropipette to replace the electrode solution (ES). To ensure complete replacement of the ES, the tip of perfusing syringe was placed close to the tip of recording micropipette (<200 µm in distance), and the volume of the new ES was 10 times greater than the volume of micropipette. Using this technique, we were able to deliver cAMP successfully into the pipette by replacing the original WCES with a WCES containing cAMP. This maneuver suppressed both inward and outward currents (Fig. 1, B and C). Removal of cAMP by subsequent perfusion with a new WCES restored these currents (Fig. 1D). These manipulations did not alter the membrane-pipette seal, which was verified by perfusing the bath with the channel blocker Gd3+ (Fig. 1E) (6, 7). In some experiments, we successfully perfused the cell interior with six different ESs. We completely replaced the ES in 2-4 min and started our recording ~10 min after each ES was delivered.


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Fig. 2.   Recording setup for patch clamping and micropipette perfusion. RC, recording chamber; MP, micropipette; MPH, micropipette holder; EH, electrode holder; M, micromanipulator; SP/SC, suction port and suction cap, respectively; ST, suction tubing connected to SC; HS, head stage of Axopatch amplifier; GE, ground electrode connected to amplifier.

Measurement of [Ca2+]i

[Ca2+]i of cell populations was measured using fura 2 as previously described (30). After cells (5-10 × 106/ml) were loaded with the acetoxymethyl ester of fura 2 and washed, they were resuspended (2-8 × 106/ml) and preincubated in cell medium with 0.5 mM CaCl2, 0.5 mM MgSO4, and 0.2% BSA with or without forskolin (10-5 M) at room temperature for 10-20 min. [Ca2+]i was assessed for at least 5 min in the fluorometer before the addition of 1.5 mM CaCl2 from a 50 mM CaCl2 stock solution to reach a final [Ca2+]o of 2.0 mM. Fluorescence was monitored for 5-10 min after the addition of Ca2+. Peak and sustained [Ca2+]i was determined as previously described (21, 30).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intracellular cAMP Blocks Im and Cm Induced by Raising [Ca2+]o

Because cAMP-dependent phosphorylation regulates Ca2+-channel activity in other cells (10, 13, 34, 35), we tested the effects of cAMP on parathyroid cell currents. Perfusion of the cell interior with a WCES that contained cAMP (3 × 10-4 M) suppressed whole cell currents significantly (Fig. 1, B and C). This effect of cAMP was reversed when the ES was changed to WCES (Fig. 1D). Both inward Ca2+ and outward Cs+ currents were blocked (>95%) by the channel blocker Gd3+ (Fig. 1E).

We next assessed the effects of perfusing the cell with ES containing different concentrations of cAMP (3 × 10-9 to 3 × 10-4 M). cAMP dose dependently suppressed the baseline Im and Cm at 0.7 mM Ca2+ and blocked the increase in Cm observed with raising [Ca2+]o from 0.7 to 90 mM (Fig. 3, A and B, and data not shown).


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Fig. 3.   Dose response of cAMP on high-extracellular Ca2+ concentration ([Ca2+]o)-induced increases in membrane current (Im) and membrane conductance (Cm) in parathyroid cells. A: voltage-clamping protocols are described in MATERIALS AND METHODS. In whole cell recordings of an isolated cell, pipette was perfused sequentially with WCES containing 0 (i), 3 × 10-6 (ii), 3 × 10-5 (not shown), and 3 × 10-4 M cAMP (iii). Arrows indicate zero current level. B: response of Cm to different Ca2+ concentrations in BS when ES contained WCES supplemented with various cAMP concentrations. Cm values were calculated from recordings in A and from data not shown. Similar results were obtained in 2 additional cells tested with 3 or more cAMP concentrations.

We found that 7 of 12 cells tested could respond to a maximal dose of cAMP (3 × 10-4 M), and the potency of cAMP varied among cells. In one cell, basal Im at 0.7 mM Ca2+ and high-[Ca2+]o-induced Im were suppressed >95% by 3 × 10-9 M cAMP (data not shown). In six other cells, both baseline Cm and the increase in Cm due to raising [Ca2+]o to 90 mM were maximally blocked by >95% by 3 × 10-4 M cAMP (Fig. 3B). Typically, cAMP concentrations in the pipette of 3-30 × 10-6 M significantly lowered Cm in these cells compared with control recordings without added cAMP. In three cells that were successfully perfused with multiple ES containing various cAMP concentrations, the dose dependence of the inhibition by this molecule was clearly demonstrated (Fig. 3B).

To assess the specificity of the response to cAMP, we perfused the pipette with cGMP, which regulates nonselective cation channels in other systems (15). Baseline Im and Im induced by raising [Ca2+]o were not suppressed by intracellular perfusion with 3 × 10-4 M cGMP (Fig. 4, A-C, and data not shown). These currents could be blocked (approx 70%) by the subsequent perfusion with a WCES containing 3 × 10-4 M cAMP (Fig. 4D), confirming the specificity of the response to cAMP. Similar results were observed in two other cells (data not shown).


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Fig. 4.   Effects of cGMP and cAMP on Im. Cell was bathed in either 0.7 mM Ca-10 mM TEA (A) or 90 mM Ca-10 mM TEA acetate BS (B-D). Recording pipette was first filled with WCES (A and B), which was replaced sequentially with WCES containing cGMP (3 × 10-4 M; WCES+cGMP; C) and then WCES containing cAMP (3 × 10-4 M; WCES+cAMP; D). Arrows indicate zero current level. This experiment is representative of results observed in 3 cells.

We next assessed whether the effects of cAMP were dependent on phosphorylation and whether they were reversible. Whole cell currents were recorded from isolated parathyroid cells perfused sequentially with the following pipette solutions: 1) WCES, 2) WCES+cAMP, 3) NRS-free WCES+cAMP, and 4) WCES+cAMP. Initial recordings were made in a BS containing 0.7 mM Ca2+. Before pipette solutions were changed, the BS was changed to vary [Ca2+]o stepwise from 0.7 to 90 mM Ca2+ to assess the effects of different pipette constituents on the responsiveness of the cell to raising [Ca2+]o.

When the pipette solution contained cAMP (3 × 10-3 M; see Fig. 5Aii), Im and Cm were markedly suppressed compared with controls as was the response to perfusion of the bath with high [Ca2+]o (Fig. 5, Ai, Aii, and B). When the NRS and ATP were removed from the pipette solution (Fig. 5, Aiii and B), the inhibition by cAMP of Im and Cm was reversed, and the responsiveness of Im to high [Ca2+]o was restored. When the next ES containing the NRS, ATP, and cAMP was perfused (Fig. 5Aiv), the ability of cAMP to block the baseline Im at 0.7 mM Ca2+ and the response to high [Ca2+]o were again observed (Fig. 5, A and B). Addition of ATP and the NRS (which regenerates ATP) back to the WCES restores the ability of cAMP to block Im and lower Cm (Fig. 5, Aiv and B). The inhibitory effects of cAMP, therefore, require ATP.


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Fig. 5.   Effects of intracellular perfusion of cAMP on high-[Ca2+]o-induced Im and Cm are blocked by removing nucleotide-regenerating system (NRS) and ATP from pipette perfusion solution. A: whole cell currents were recorded from a cell in which pipette was perfused with a series of solutions (in order): WCES (i), WCES+cAMP (3 × 10-4 M; ii), NRS-free WCES+cAMP (3 × 10-4 M; iii), and WCES+cAMP (3 × 10-4 M; iv). After each change in pipette solution, cell was exposed to BS containing different calcium acetate concentrations, from 0.7 to 90 mM. Arrows indicate zero current level. B: response of Cm to various Ca2+ concentrations in bath when pipette solutions were as specified above. Cm values were calculated from recordings in A and from data not shown. These data are representative of results in 5 other cells.

Perfusion of the Pipette With PKA-CS Mimics the Effects of cAMP on Membrane Currents

Because cAMP activates PKA and because the ability of cAMP to lower Im and Cm in parathyroid cells depends on the presence of ATP, we examined whether perfusion of the patch pipette with the PKA-CS mimicked the effects of cAMP in whole cell recordings. In the experiment shown in Fig. 6 and in three other cells, cAMP reversibly suppressed Im and Cm both at 0.7 mM Ca2+ (by >50%) and after raising [Ca2+]o (by >80%) (Fig. 6, Ai-Aiii and B). Perfusion of the same cell internally with a WCES supplemented with PKA-CS (5 × 10-5 g/ml) also suppressed Im and Cm at 0.7 mM Ca2+ (Fig. 6B and data not shown) and blocked the response to high [Ca2+]o in the BS (Fig. 6B). The inhibitions by cAMP and PKA-CS were comparable, ~80%. The effects of PKA-CS depended on the presence of NRS and ATP in the pipette solution, since the removal of both components from WCES blocked the inhibition of Im by PKA-CS (Fig. 6, Av and B). PKA-CS was effective at concentrations of 5 × 10-7 g/ml and was maximal at 5 × 10-5 g/ml (data not shown).


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Fig. 6.   Catalytic subunit of protein kinase A (PKA-CS) mimicked effects of intracellular perfusion with cAMP on high-[Ca2+]o-induced increases in Im and Cm. A: currents were recorded from a cell perfused internally with a series of ESs (in order): WCES (i), WCES+cAMP (3 × 10-4 M; ii), WCES (rinsed) (iii), WCES+PKA-CS (5 × 10-5 g/ml; iv), NRS-free WCES+PKA-CS (5 × 10-5 g/ml; v), and same as in v, but at end of experiment, Gd3+ (3 × 10-3 M) was added to bath containing 45 mM calcium acetate to verify integrity of electric seal (vi). With each change in ES, recordings were obtained in a series of BSs containing different calcium acetate concentrations. Only current traces recorded in 45 mM Ca-10 mM TEA acetate BS are presented in A. Arrows indicate zero current level. B: response of Cm to different Ca2+ concentrations in bath when pipette was perfused with ES specified. Cm values were calculated from recordings in A and from data not shown for other Ca2+ concentrations. These data are representative of experiments in 4 cells.

We next determined whether either the NRS, ATP, or both were required for the inhibition by PKA-CS. As shown in Fig. 7, pipette perfusion with WCES plus PKA-CS again dramatically lowered Im (Fig. 7, A and B). When the NRS and ATP were deleted from the pipette solution, there was no inhibition by PKA-CS (Fig. 7C). Instead, the addition of ATP alone back to the pipette solution restored the ability of the PKA-CS to inhibit membrane currents (Fig. 7D and data not shown). The NRS was not essential for the inhibition of membrane currents by PKA-CS in parathyroid cells.


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Fig. 7.   Suppression of high-[Ca2+]o-induced increases in Im by intracellular perfusion of PKA-CS depends on presence of ATP. Whole cell currents were recorded from a parathyroid cell in 90 mM Ca-10 mM TEA acetate BS with a pipette that was sequentially perfused with WCES (A), WCES+PKA-CS (5 × 10-5 g/ml; B), NRS-free WCES+PKA-CS (5 × 10-5 g/ml; C), and NRS-free WCES+PKA-CS (5 × 10-5 g/ml) plus MgATP (4 × 10-3 M) (D). Arrows indicate zero current level. These tracings are representative of similar results in 3 cells.

Effects of Forskolin on cAMP Content and Whole Cell Membrane Currents in Parathyroid Cells

We next tested whether manipulations that stimulate increases in intracellular cAMP reproduce the effects of perfusion of the cell interior with cAMP. Voltage-clamped parathyroid cells were perfused with a BS containing the adenylate cyclase activator forskolin. Forskolin at concentrations >= 10-5 M increased cAMP levels in intact cells by at least 25 ± 3-fold (P < 0.001 vs. controls, n = 15 experiments from 2 cell preparations) compared with basal levels of approx 0.1 pmol/106 cells. Under whole cell recording conditions, forskolin (10-4 M) in the BS suppressed Im at 90 mM Ca2+ by ~50% (Fig. 8, A and B). The inhibition by forskolin depended on the presence of ATP in the BS, again supporting the involvement of protein phosphorylation in this effect (Fig. 8, C and D). Similar findings were seen in two other cells.


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Fig. 8.   Suppression of high-[Ca2+]o-induced increases in Im by forskolin is ATP dependent. Whole cell recordings from a parathyroid cell exposed to BS (90 mM Ca-10 mM TEA acetate BS) without (A) or with (B-D) forskolin (10-4 M). Pipette was first filled with WCES (A and B), which was subsequently replaced by NRS-free WCES (C) and NRS-free WCES+MgATP (4 × 10-3 M; D). Compositions of ES and BS are noted as ratio of ES to BS (ES/BS). Arrows indicate zero current level. Similar results were obtained in 3 cells.

Effects of cAMP on Nifedipine- and Cd2+-Resistant Currents in Parathyroid Cells

We previously reported that nonselective cation currents in parathyroid cells have two major components (6). Type I currents are blocked by Cd2+ and nifedipine and increase with raising of [Ca2+]o. Type II currents, in contrast, are not activated by raising [Ca2+]o and are Cd2+ and nifedipine insensitive. The present studies support the idea that cAMP, PKA-CS, and forskolin mainly affect type I currents. To examine whether these agents could also modulate type II currents, we recorded from cells in a BS containing 3 mM Cd2+. Cd2+ blocks type I currents (>95%) but not type II currents (6). In the presence of a Cd2+-containing BS, intracellular perfusion of cAMP (3 × 10-4 M) did not alter Im (Fig. 9A) or Cm (Fig. 9B) at either low or high [Ca2+]o. Thus it appears that cAMP mainly inhibits high-[Ca2+]o-activated type I currents in parathyroid cells.


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Fig. 9.   Type II Ca2+-conducting currents in parathyroid cells are not affected by intracellular perfusion of cAMP. A: currents were recorded from a cell in a bath perfused with different calcium acetate concentrations (0.7-90 mM) in presence of Cd2+ (3 × 10-3 M) to block type I currents. Recording pipette was first filled with WCES, which was then replaced by WCES+cAMP (3× 10-4 M). Gd3+ (3 × 10-3 M) was added to bath to verify integrity of electric seal at end of experiment. Arrows indicate zero current level. B: effects of perfusing pipette with WCES+cAMP (3 × 10-4 M) and perfusing bath with Cd2+ (3 × 10-3 M) on Cm at different [Ca2+]o values. Compositions of ES and BS in different recordings are noted as ES/BS.

Effects of Forskolin on [Ca2+]i in Parathyroid Cells

Because type I currents are activated by high [Ca2+]o and inhibited by forskolin, we examined the ability of forskolin to suppress the high-[Ca2+]o-induced increases in [Ca2+]i. As shown in Fig. 10, preincubation of parathyroid cells without or with forskolin (10-5 M) had no significant effects on [Ca2+]i at 0.5 mM [Ca2+]o or either the transient or the sustained increases in [Ca2+]i due to the raising of [Ca2+]o to 2.0 mM. Thus forskolin-induced changes in cAMP do not detectably alter Ca2+ mobilization over this range of [Ca2+]o.


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Fig. 10.   Effects of forskolin on intracellular Ca2+ concentration ([Ca2+]i) in parathyroid cells. [Ca2+]i at 0.5 mM Ca2+ was assessed in cells incubated for 10-20 min with (+; 10-5 M) or without (-) forskolin. [Ca2+]o was then raised to 2.0 mM, and both immediate and sustained increases in [Ca2+]i were monitored. These results are from 12 experiments done on 3 independent cell preparations.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PTH plays an important role in the maintenance of systemic Ca2+ homeostasis. Changes in [Ca2+]o regulate the rate of PTH release and biosynthesis (2, 25). [Ca2+]o affects these changes by interacting with parathyroid Ca2+-sensing receptors (CaRs), which couple changes in [Ca2+]o to sustained increases in [Ca2+]i and other second messengers. Increases in [Ca2+]i are likely due to mobilization of Ca2+ from intracellular stores and Ca2+ influx across the membrane. Previous studies have implicated dihydropyridine-sensitive Ca2+ channels in both Ca2+ influx and the regulation of PTH secretion (26). The expression of such channels has been inferred on the basis of functional studies with pharmacological probes and antibodies against Ca2+-channel subunits (12, 18). Little direct evidence exists, however, regarding the nature of the Ca2+ influx mechanism(s) activated by high [Ca2+]o in these cells.

We have previously identified and characterized voltage-independent and dihydropyridine-sensitive cation currents by whole cell patch clamping. These currents may provide a mechanism for Ca2+ influx in parathyroid cells (6). In the present study, we further demonstrate by several lines of evidence that these currents, which are responsive to changes in [Ca2+]o, are regulated by cAMP-dependent phosphorylation.

Phosphorylation is a common mechanism for regulating Ca2+ channels. In both cardiac and skeletal muscle cells, PKA-mediated phosphorylation activates Ca2+ channels and thereby enhances Ca2+ influx (5, 31). In contrast, cAMP-dependent phosphorylation blocks voltage-dependent L-type Ca2+ channels in vascular smooth muscle cells (35). We found that cAMP and PKA-CS, through ATP-dependent mechanisms, suppress high-[Ca2+]o-induced, dihydropyridine-sensitive Ca2+ currents in parathyroid cells. Thus the nature of the regulation of channel activity by cAMP varies depending on the cell type.

The marked suppression of membrane currents by cAMP in the perfusion pipette was dose dependent and reversible. These effects are likely to be specific for several reasons. 1) They were detectable at concentrations of 3 × 10-9 to 3 × 10-5 M cAMP in the pipette solution. This concentration range is comparable to those at which cAMP activates PKA in vitro (10-9 to 10-5 M) (33). 2) cGMP did not reproduce these effects. 3) The effects observed with cAMP were also observed when the PKA-CS was in the perfusion pipette or when the bath was perfused with forskolin. Thus manipulations that would either increase intracellular cAMP content or enhance cAMP-dependent phosphorylation produced the same inhibition of the nonselective currents we monitored.

There are a variety of mechanisms by which cAMP- and PKA-mediated phosphorylation can regulate channel currents. Cyclic nucleotides can modify the gating properties of channels directly by binding to the channel protein (10, 13, 36, 37) or indirectly by modifying the channel protein through cAMP- or cGMP-dependent phosphorylation (15, 24, 32, 34).

The effect of cAMP on high-[Ca2+]o-induced Ca2+ currents in parathyroid cells is likely through a pathway involving phosphorylation. This is based on the dependence of the effect of cAMP on the presence of ATP. There are many candidate substrates for cAMP-dependent phosphorylation in the parathyroid that could modulate ion channel activity. These include the channel subunits themselves, other channel regulators, and even the CaRs. Any of these molecules could be affected by cAMP-dependent phosphorylation that could then alter the activation of membrane currents. The CaR may be a less likely target of PKA-mediated phosphorylation, since the bovine parathyroid CaR lacks consensus sites for PKA-mediated phosphorylation (3). It has been shown, however, that the alpha 1- and beta -subunits of dihydropyridine-sensitive Ca2+ channels are targets of cAMP-dependent phosphorylation in other systems (9, 14, 20, 27). Therefore, it is a real possibility by analogy that cAMP-dependent phosphorylation suppresses membrane currents in the parathyroid via the phosphorylation of a channel protein. Studies in Xenopus laevis oocytes injected with mRNA encoding cardiac Ca2+-channel subunits suggest that phosphorylation of other as yet unidentified proteins can also be responsible for the suppression of channel activity (8). Detailed understanding of the mechanism by which cAMP and PKA-CS regulate type I nonselective cation currents in parathyroid cells awaits the molecular identification of the channel protein(s) responsible for this conductance.

Our studies showed that internal perfusion of parathyroid cells with cAMP suppressed Ca2+-conducting currents, presumably through the activation of PKA. Therefore, in the whole cell configuration, these cells clearly have endogenous PKA activity. This idea may explain our observations that removal of the NRS and ATP not only restored the Im and Cm to levels recorded before addition of cAMP but often enhanced Im and Cm slightly. In addition, switching the ES directly from the normal WCES to an NRS-free WCES increased both basal and high-[Ca2+]0-induced Im and Cm. One possible explanation for this enhancement is that endogenous cAMP may be involved in setting the baseline Im and Cm in parathyroid cells.

In our studies, not all cells were responsive to patch pipette perfusion with cAMP. This may be due to inherent heterogeneity in the cell population in terms of their expression of the relevant effector proteins, either channel subunits or other channel regulators. In previous studies, we (6) and others (22, 23) have observed that there is heterogeneity in the intracellular Ca2+ responses of parathyroid cells exposed to the changes in the [Ca2+]o. Alternatively, our pipette perfusion techniques may alter either the responses or the viability of less robust cells.

The inhibition of Im by perfusion of the cell interior with cAMP or the PKA-CS may explain previous observations regarding high-[Ca2+]o-mediated signal transduction in parathyroid cells. It is known that high [Ca2+]o decreases forskolin-stimulated cAMP content (28) and increases [Ca2+]i in these cells (2, 25). Results of the present study indicate that an increase in intracellular cAMP is associated with inhibition of cation influx. Thus the decrease in cAMP content due to high [Ca2+]o might be predicted to promote cation (and possibly Ca2+) influx in parathyroid cells. By this line of reasoning, the reduction in cAMP induced by high [Ca2+]o could contribute to cellular Ca2+ mobilization and the inhibition of PTH secretion. This hypothesis is supported by observations that forskolin (28) and other agents that raise cAMP content (2) increase PTH secretion in intact parathyroid cells. Whether the increase in PTH secretion due to forskolin is related to the suppression of the cation currents we studied or to other mechanisms is unknown.

Arguing against the paradigm that changes in Ca2+ mobilization mediate the effects of forskolin on PTH secretion are our measurements of [Ca2+]i. These determinations fail to show an effect of forskolin on this parameter. There are several explanations, however, for differing results between patch-clamp recordings and intracellular Ca2+ determinations. 1) Patch clamping is inherently a more sensitive, direct, and better controlled way to detect changes in ion flux than is the measurement of steady state [Ca2+]i. 2) Recordings from single cells selected on the basis of responsiveness to cAMP allowed us to study only those cells with this response in the population. Fluorescence determinations from a cell population are inherently less sensitive to changes that may be occurring in a subpopulation, particularly if the changes are small. 3) The levels of critical intracellular regulators may differ under the two different experimental conditions. In whole cell recordings, cells were perfused with a solution of defined composition. A continuous supply of ATP, GTP, and an NRS was provided. In contrast, [Ca2+]i was determined in intact cells under more physiological conditions. Under these conditions, cells may maintain different basal levels of cyclic nucleotides, ATP, and GTP than those we provided in whole cell recordings. Furthermore, these levels may vary during the course of the experiments. 4) The electrophysiological conditions we used favor isolation of and recording from nonselective cation currents, for which Ca2+ and other cations can be the charge carriers. We blocked K+ currents with TEA+ and Cs+ (6), and Cl- was omitted from our bath and pipette solutions. In contrast, determinations of [Ca2+]i are difficult to make under such rigidly controlled conditions. For all of the above reasons, differing intracellular Ca2+ and electrophysiological results are likely due to inherent differences in the two assay systems.

The role of these nonselective cation currents in the overall function of the parathyroid cell is unknown. Our previous studies suggested that these currents might play a role in intracellular Ca2+ mobilization, since Gd3+, which blocks these currents, also reduced [Ca2+]i (6). The present study, however, does not implicate a role for the type I nonselective cation conductance, which is responsive to high [Ca2+]o, in the determination of [Ca2+]i, at least under the conditions employed in this study. Whether these cation currents are conducted by Ca2+ or another cation(s) under physiological circumstances is unknown. These issues, as well as the functional role played by this current in parathyroid physiology, await the molecular identification of the responsible channel protein. This will greatly facilitate an investigation into the role of this current in extracellular Ca2+-regulated parathyroid function.

    ACKNOWLEDGEMENTS

These studies were supported by National Institutes of Health Grant R01-43400, a Merit Review from the Research Service, Department of Veterans Affairs, and the Northern California Chapter of the Arthritis Foundation. D. Shoback was a Clinical Investigator of the Department of Veterans Affairs during these studies.

    FOOTNOTES

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. §1734 solely to indicate this fact.

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

Received 3 February 1998; accepted in final form 24 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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