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First published online December 2, 2005
Journal of Experimental Biology 208, 4747-4756 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01967

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Biophysical properties of voltage-gated Na⁺ channels in frog parathyroid cells and their modulation by cannabinoids

Yukio Okada^1,*, Kotapola G. Imendra⁴, Toshihiro Miyazaki², Hitoshi Hotokezaka³, Rie Fujiyama¹, Jorge L. Zeredo¹, Takenori Miyamoto⁵ and Kazuo Toda¹

¹ Integrative Sensory Physiology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
² Oral Cytology and Cell Biology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
³ Orthodontics and Biomedical Engineering, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Nagasaki 852-8588, Japan
⁴ Department of Physiology, Faculty of Medicine, University of Rhuna, Galle, Sri Lanka
⁵ Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Bunkyo-ku, Tokyo 112-8681, Japan

^* Author for correspondence (e-mail: okada{at}net.nagasaki-u.ac.jp)

Accepted 2 November 2005

	Summary

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The membrane properties of isolated frog parathyroid cells were studied using perforated and conventional whole-cell patch-clamp techniques. Frog parathyroid cells displayed transient inward currents in response to depolarizing pulses from a holding potential of –84 mV. We analyzed the biophysical properties of the inward currents. The inward currents disappeared by the replacement of external Na⁺ with NMDG⁺ and were reversibly inhibited by 3 µmol l^–1 TTX, indicating that the currents occur through the TTX-sensitive voltage-gated Na⁺ channels. Current density elicited by a voltage step from –84 mV to –24 mV was –80 pA pF^–1 in perforated mode and –55 pA pF^–1 in conventional mode. Current density was decreased to –12 pA pF^–1 by internal GTPS (0.5 mmol l^–1), but not affected by internal GDPßS (1 mmol l^-1). The voltage of half-maximum (V_1/2) activation was –46 mV in both perforated and conventional modes. V_1/2 of inactivation was –80 mV in perforated mode and –86 mV in conventional mode. Internal GTPS (0.5 mmol l^–1) shifted the V_1/2 for activation to –36 mV and for inactivation to –98 mV. A putative endocannabinoid, 2-arachidonoylglycerol ether (2-AG ether, 50 µmol l^–1) and a cannabinomimetic aminoalkylindole, WIN 55,212-2 (10 µmol l^–1) also greatly reduced the Na⁺ current and shifted the V_1/2 for activation and inactivation. The results suggest that the Na⁺ currents in frog parathyroid cells can be modulated by cannabinoids via a G protein-dependent mechanism.

Key words: parathyroid, voltage-gated Na⁺ channel, G protein, activation, inactivation, cannabinoid, frog

	Introduction

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Parathyroid hormone (PTH) regulates extracellular free Ca²⁺ concentration ([Ca²⁺]_o) in cooperation with 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) and calcitonin (CT). On the other hand, [Ca²⁺]_o regulates the secretion of PTH from parathyroid cells through extracellular Ca²⁺-sensing receptor (CaR; Brown et al., 1993; Hofer and Brown, 2003). High [Ca²⁺]_o inhibits and low [Ca²⁺]_o enhances PTH secretion. It is believed that extracellular Ca²⁺ inhibits the secretion of PTH via the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). However, the molecular mechanism by which [Ca²⁺]_i regulates PTH secretion is not well elucidated.

Several electrophysiological studies have been performed in mammalian parathyroid cells. Those using classical intracellular microelectrodes indicated that rodent parathyroid cells display a deep resting potential (about –70 mV), which is depolarized by increasing [Ca²⁺]_o (Bruce and Anderson, 1979; López-Barneo and Armstrong, 1983). Later, the patch-clamp technique was applied on bovine, human and rodent parathyroid cells (Castellano et al., 1987; Jia et al., 1988; Komwatana et al., 1994; Kanazirska et al., 1995; McHenry et al., 1998; Välimäki et al., 2003). These studies showed that parathyroid cells possess some types of K⁺ channels. Other studies suggested the presence of voltage-gated Ca²⁺ channels in bovine and goat parathyroid cells (Sand et al., 1981; Chang et al., 2001). However, voltage-gated Na⁺ channels could not be found in any of the aforementioned studies.

Ion channels are regulated by neurotransmitters and hormones via G protein-coupled receptors (GPCRs; Wickman and Clapham, 1995; Dascal, 2001). GPCRs dissociate heterotrimeric G proteins (Gß) to G-GTP and Gß. Both subunits can regulate a variety of ion channels directly (via physical interactions between G protein subunits and the channel protein) or indirectly (via second messengers and protein kinases).

In the present study, we report that frog parathyroid cells possess voltage-gated Na⁺ channels and that their activity may be modulated by cannabinoids.

	Materials and methods

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Cell preparation
Adult bullfrogs Rana catesbeiana Shaw weighing 250–550 g were used for the experiment over the course of a year. Experiments were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University. Parathyroid cells were isolated from the parathyroid glands of decapitated and pithed animals. Two pairs of the oval parathyroid glands in both sides were quickly dissected from the pre-cardial region, where they lie near the ventral branchial bodies and attached to the carotid arteries (Sasayama and Oguro, 1974). The glands were cut into small pieces in Ca²⁺-free saline containing 2 mmol l^–1 EDTA and incubated for 12–15 min in 2 ml of the same saline containing 10 mmol l^–1 L-cysteine and 10 units ml^–1 papain (Sigma, St Louis, MO, USA). The glands were then rinsed with normal saline. The individual cells were dissociated by gentle trituration in normal saline. Isolated parathyroid cells displayed an oval-shape, with a diameter of about 10 µm (Fig. 1).

View larger version (90K): [in this window] [in a new window]   Fig. 1. Freshly isolated frog parathyroid cells are white and oval-shaped in this phase-contrast image. Two large cells in the center of the image are the erythrocytes. Bar, 50 µm.

Data analysis
Data were analyzed with pCLAMP and Origin 7.5 (Origin Lab, Northampton, MA, USA). Unless stated otherwise, the data are presented as means ± S.E.M., significance was tested by Student's t-test and a difference was considered significant if P<0.05. The steady-state inactivation curves were fitted with a Boltzmann equation as follows:

(1)

where I/I_max is the current magnitude normalized to its maximum value, V designates the conditioning voltage, V_1/2 is the voltage at which the magnitude is half-maximum, and k is a Boltzmann slope factor, which reflects the voltage sensitivity of steady-state inactivation of the current. The conductance for activation was estimated according to the following equation:

(2)

where G is the peak conductance, I is the current magnitude at the test voltage (V), and V_rev is the apparent reversal potential for the Na⁺ current. The activation curve was also fitted with the Boltzmann equation as follows:

(3)

where G/G_max is the conductance magnitude normalized to its maximum value, V designates the test voltage, and other parameters are the same as above. Recovery from inactivation was fitted using:

(4)

where t is time, a is maximum magnitude, and is time constant for the recovery.

Solution and drugs
Normal saline solution consisted of (in mmol l^–1): NaCl 115, KCl 2.5, CaCl₂ 1.8, Hepes 10; glucose 20, pH 7.2. The pH of normal saline and other solutions was adjusted by Tris base. The extracellular Na⁺-free solution was prepared by the replacement of Na⁺ with NMDG⁺. The solution exchange was done by gravity flow. For stock solution, tetrodotoxin (3 mmol l^–1; Sigma), spermine tetrahydrochloride (100 mmol l^–1; Sigma) and adrenaline bitartrate (10 mmol l^–1; Sigma) were dissolved in deionized water. WIN 55,212-2 mesylate (10 mmol l^–1; Tocris, Bristol, UK), phorbol 12,13-dibutyrate (PDBu, 10 mmol l^–1; Sigma), forskolin (10 mmol l^–1; Sigma) and chelerythrine chloride (10 mmol l^–1; Sigma) were dissolved in dimethylsulphoxide (DMSO). 2-arachidonoylglycerol ether (2-AG ether, 13.7 mmol l^–1) dissolved in ethanol was purchased from Tocris. Samples of the stock solutions were added to normal saline solution to give the desired final concentrations.

The standard K⁺ internal solution contained (in mmol l^–1): KCl 100, CaCl₂ 0.1, MgCl₂ 2, EGTA 1, Hepes 10, pH 7.2. GTPS (0.5 mmol l^–1, Sigma) and GDPßS (1 mmol l^–1, Sigma) were dissolved in the internal solution on every experimental day.

All experiments were carried out at room temperature (20–25°C).

	Results

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Basal properties of isolated frog parathyroid cells
Under conventional whole-cell mode in a standard K⁺ internal solution, frog parathyroid cells displayed resting potential of –10 to –70 mV (–30±2 mV, N=44). The input resistance ranged from 4.7 to 25.0 G (13.7±0.9 G, N=44) and the membrane capacitance was 5.8–11.0 pF (7.6±0.2 pF, N=44). On the other hand, under perforated whole-cell mode using amphotericin B, the cell displayed resting potentials of –10 to –87 mV (–26±3 mV, N=26). In perforated mode, the input resistance ranged from 4.5 to 50.0 G (11.1±1.8 G, N=26), and the membrane capacitance was 6.0–13.0 pF (7.9±0.4 pF, N=26). Differences in basal membrane properties were not significant between conventional and perforated conditions.

Activation
After attaining the perforated whole-cell configuration in normal saline solution, almost all frog parathyroid cells displayed transient inward currents in response to depolarizing voltage steps from a holding potential of –84 mV (Fig. 2A). Although the pipette contained a K⁺ internal solution, leak currents, but not the voltage-gated outward current could be found. The threshold for the inward current activation ranged from –64 to –54 mV (Fig. 2B). The inward currents were activated more rapidly with successive depolarizing steps. The activation time reaching a peak at –24 mV, for instance, was 0.69±0.04 ms (N=27). Removal of external Na⁺ completely abolished the inward currents (3 cells; Fig. 3A), indicating that the currents were caused predominately by Na⁺. Application of 3 µmol l^–1 TTX (a voltage-gated Na⁺ channel blocker) totally eliminated the inward currents (8 cells; Fig. 3B) after about 4 min. The currents recovered to 92.2±12.2% (N=8) of the controls after about 10 min of washing with normal saline solution.

View larger version (13K): [in this window] [in a new window]   Fig. 2. A representative example of perforated whole-cell current of a frog parathyroid cell in normal saline solution. (A) Transient inward currents were elicited in response to 15 ms voltage steps between –74 to +56 mV in 10 mV increments from a holding potential of –84 mV. The leak currents were not subtracted from the current traces. (B) Pooled current–voltage (I–V) relationships for the inward currents (N=17) elicited by the voltage steps. Values are means ± S.E.M.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effects of the elimination of external Na+ (A) and the addition of 3 µmol l–1 TTX to external normal saline solution (B) on the transient inward currents. The currents were elicited by 15 ms step pulses from a holding potential of –84 mV to a test potential of –24 mV.

Membrane-delimited G protein-regulation of voltage-gated Na⁺ channels has been suggested. We also investigated the effect of GTPS on the Na⁺ channels in frog parathyroid cells. Activation parameters were compared with each other in the presence and absence of GTPS. Peak inward current elicited by a voltage step from –84 to –24 mV was –663±74 pA (N=27) in perforated mode, whereas the current decreased to –419±33 pA (N=30, P<0.05) in conventional mode. GDPßS (1 mmol l^–1) added to the intracellular solution did not affect the current amplitude (–413±88 pA, N=7) as compared with conventional mode, but internal 0.5 mmol l^–1 GTPS significantly decreased the current to –93±10 pA (N=8, P<0.05). Similar results were observed for current density (expressed as the ratio of current amplitude to cell capacitance). The magnitudes of the current density were –79.5±6.6 pA pF^–1 (N=27) in perforated mode, –55.2±4.4 pA pF^–1 (N=30) in conventional mode, –49.8±9.2 pA pF^–1 (N=7) in the condition containing 1 mmol l^–1 GDPßS and –11.6±1.3 pA pF^–1 (N=8) in the condition containing 0.5 mmol l^–1 GTPS, respectively. The voltage of half-maximum activation V_1/2 in perforated mode was estimated from the activation curve (V_1/2=–45.7±0.8 mV, N=17; Fig. 4A). The V_1/2 in conventional mode (–46.1±1.1 mV, N=18) was almost the same. Addition of 1 mmol l^–1 GDPßS into the pipette solution did not change the V_1/2 (–45.1±1.7 mV, N=6) as compared with conventional mode, but internal 0.5 mmol l^–1 GTPS shifted the V_1/2 to –35.5±3.3 mV (N=8, P<0.05; Fig. 4B).

View larger version (12K): [in this window] [in a new window]   Fig. 4. Voltage dependence of activation of the Na+ currents, determined by I–V relationships for the Na+ currents elicited by the voltage steps. Smooth curves are simple Boltzmann functions. (A) V1/2=–45.7 mV, k=–6.4 mV for perforated recording (filled circles), V1/2=–46.1 mV, k=–5.2 mV for conventional recording (open circles). (B) V1/2=–35.5 mV, k=–7.6 mV for 0.5 mmol l–1 GTPS (filled triangles), V1/2=–45.1 mV, k=–4.4 mV for 1 mmol l–1 GDPßS (open triangles). For comparison, the broken line for conventional recording is plotted in B. The values are means ± S.E.M. obtained from 6–18 cells.

Inactivation
We measured inactivation time constant () for inactivation onset during a test pulse to –24 mV. The inactivation time constant in perforated mode (0.87±0.08 ms, N=27) was shorter than the constant in conventional mode (1.13±0.06 ms, N=30, P<0.05). Internal 1 mmol l^–1 GDPßS and 0.5 mmol l^–1 GTPS did not significantly change the constant as compared with conventional mode (data not shown).

The voltage-dependence of steady-state inactivation was studied in a conventional manner. As the level of the conditioning pulses was shifted to the depolarizing direction, the magnitude of the inward currents decreased (Fig. 5A). The point of the half-maximum inactivation (V_1/2) was –79.8±2.1 mV (N=14) in perforated mode (Fig. 5B). The V_1/2 in conventional mode shifted to hyperpolarizing direction (–86.3±2.0 mV, N=12, P<0.05). Internal 1 mmol l^–1 GDPßS did not affect the V_1/2 (–86.1±3.0 mV, N=7) as compared with conventional mode, but internal 0.5 mmol l^–1 GTPS significantly shifted the V_1/2 to –98.4±2.1 mV (N=12, P<0.05; Fig. 5C).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Voltage dependence of steady-state inactivation of the Na+ currents. (A) The dependence was determined by measuring peak current elicited by a single depolarization to –34 mV from a range of 200 ms conditioning voltages. (B) V1/2=–79.8 mV, k=8.2 mV for perforated recording (filled circles), V1/2=–86.3 mV, k=9.4 mV for conventional recording (open circles). (C) V1//2=–98.4 mV, k=9.6 mV for 0.5 mmol l-1 GTPS (filled triangles), V1/2=–86.1 mV, k=10.0 mV for 1 mmol l–1 GDPßS (open triangles). For comparison, the broken line for conventional recording is plotted in B. The values are means ± S.E.M. obtained from 7–14 cells.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Time course of recovery from inactivation. (A) Inward currents recorded using a double pulse protocol in which a 20 ms control pulse from –84 to –24 mV was followed by a second identical voltage pulse. (B) The plots show the recovery time course of the Na+ currents. The curves represent the fits of a single exponential function, giving values of time constants of 9.6 ±1.2 ms (N=5) for perforated mode (filled circles), 13.1±0.7 ms (N=5) in conventional mode (open circles) and 16.5±2.4 ms (N=5) for the condition containing 0.5 mmol l–1 GTPS in the internal solution (open triangles).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effects of WIN 55,212-2 and 2-AG ether on the Na+ currents. (A) Reversible inhibition of the Na+ current by 10 µmol l–1 WIN 55,212-2. The currents were elicited by a 20 ms pulse from a holding potential of –84 mV to a test potential of –24 mV. The current traces labelled a–c were obtained at the times indicated by the same letter on the time course. (B) Representative example of time course of the current signal. (C) Na+ current magnitudes before and after superfusion with each drug. Values are means ± S.E.M. Numerals within parentheses are number of the cells sampled.

View larger version (33K): [in this window] [in a new window]   Fig. 8. Effects of WIN 55,212-2 and 2-AG ether on activation and inactivation of the Na+ currents. (A) Voltage dependence of activation before and after superfusion with 10 µmol l–1 WIN 55,212-2. V1/2=–48.6 mV, k=–5.1 mV for control (open squares), V1/2=–37.0 mV, k=–5.0 mV for WIN 55,212-2 (filled circles). (B) Voltage dependence of inactivation before and after superfusion with 10 µmol l–1 WIN 55,212-2 (filled circles). V1/2=–76.9 mV, k=8.1 mV for control (open squares), V1/2=–94.4 mV, k=9.0 mV for WIN 55,212-2. (C) V1/2 of activation before (white bars) and after superfusion with each drug (hatched bars). (D) V1/2 of inactivation before (white bars) and after superfusion with each drug (hatched bars). Values are means ± S.E.M. Numerals within parentheses are number of the cells sampled.

Effects of protein kinases on the Na⁺ current
The cytoplasmic loop in the Na⁺ channels possesses several protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites (Cantrell et al., 2003). To determine the role of PKC on Na⁺ current, we tested the effect of a PKC activator, PDBu on the properties of Na⁺ current. PDBu (10 µmol l^–1) decreased the Na⁺ current at –24 mV to 37±8% of the controls (Fig. 9C). A wash-out with normal saline solution was not observed (Fig. 9A). PDBu did not significantly shift the V_1/2 of activation (Fig. 10A), but induced the hyperpolarizing shift of 11.9±2.6 mV (N=5, P<0.05) in the V_1/2 of inactivation (Fig. 10B). An adenylyl cyclase activator, forskolin (10 µmol l^–1) did not significantly inhibit the Na⁺ current (Fig. 9C).

View larger version (18K): [in this window] [in a new window]   Fig. 9. Effects of PDBu and forskolin on the Na+ currents. (A) Representative example of time course of the current signal after superfusion with 10 µmol l–1 PDBu. (B) Representative example of time course of the current signal after superfusion with 10 µmol l–1 forskolin. (C) Na+ current magnitude before (white bars) and after superfusion with each drug (hatched bars). The currents were elicited by 20 ms pulse from a holding potential of –84 mV to a test potential of –24 mV. Values are means ± S.E.M. Numerals within parentheses are number of the cells sampled.

View larger version (17K): [in this window] [in a new window]   Fig. 10. Effect of 10 µmol l–1 PDBu on activation and inactivation of the Na+ currents. (A) Voltage dependence of activation. V1/2=–47.7 mV, k=–5.3 mV for control (open squares), V1/2=–44.3 mV, k=–6.0 mV for PDBu (filled circles). (B) Voltage dependence of inactivation. V1/2=–81.8 mV, k=8.1 for control (open squares), V1/2=–93.7 mV, k=9.0 mV for PDBu (filled circles). Values are mean ± S.E.M. obtained from 5–8 cells.

View larger version (26K): [in this window] [in a new window]   Fig. 11. Effect of WIN 55,212-2 on the Na+ currents in the presence of an inhibitor of PKC. (A) Representative example of time course of the current signal. The currents were elicited by a pulse from a holding potential of –84 mV to a test potential of –24 mV. (B) Mean values of Na+ current magnitudes before (white bar) and after superfusion with 10 µmol l–1 chelerythrine and 10 µmol l–1 WIN 55,212-2 (hatched bar). Numerals within parentheses are number of the cells sampled. (C) Voltage dependence of activation. V1/2=–42.0 mV, k=–6.1 mV for control (open squares), V1/2=–30.7 mV, k=–8.0 mV for chelerythrine plus WIN 55,212-2 (filled circles). (D) Voltage dependence of inactivation. V1/2=–83.5 mV, k=8.4 mV for control (open squares), V1/2=–103.8 mV, k=7.6 mV for chelerythrine plus WIN 55,212-2 (filled circles). The values are mean ± S.E.M. obtained from three cells.

	Discussion

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The present study shows that frog parathyroid cells express TTX-sensitive voltage-gated Na⁺ channels in the cell membrane. However, most Na⁺ channels were silent at resting potential (–30 mV). The silent Na⁺ channels in the parathyroid cells could only be restored if a hyperpolarizing voltage was applied. Similarly, rat gonadotropes in the anterior lobe of the pituitary glands fire action potentials at the termination of the hyperpolarization elicited by gonadotropin-releasing hormone (GnRH; Tse and Hille, 1993); in these cells, fast depolarizations in action potentials open voltage-gated Ca²⁺ channels that the allow entry of extracellular Ca²⁺. Nevertheless, in the present experiments, the V_1/2 for inactivation was about –80 mV in perforated mode, and few cells that possess such a deep resting potential are excitable.

By comparing the activation and inactivation processes in the presence and absence of GTPS, we observed evidence that the activation and inactivation of Na⁺ currents are regulated by guanine nucleotides, perhaps as a G protein-coupled mechanism. In the absence (conventional) of GTPS, the mean V_1/2 for Na⁺ current activation was about –46 mV. A similar mean value was observed in the presence of GDPßS in the pipette solution. Since internal GTPS shifted the V_1/2 to a more positive level (–36 mV), the presence of GTPS results in the activation of a G protein-dependent mechanism, which may be functionally important in regulating Na⁺ current activation. A similar effect of GTPS was also observed in the inactivation process. Our results suggest that a G protein-dependent inhibitory mechanism may be involved in both activation and inactivation processes in the voltage-gated Na⁺ channels of frog parathyroid cells. Furthermore, in perforated mode, the V_1/2 for inactivation shifted to a more positive value than in conventional mode. This also suggests that the Na⁺ channels may be regulated by another enhancing mechanism. It has been reported that voltage-gated Na⁺ channels are under the regulation of G proteins. In rat cardiac myocytes, internal GTPS inhibits the Na⁺ channel by shifting the steady-state inactivation to more negative potentials (Schubert et al., 1989 and 1990). In contrast, GTPS enhances Na⁺ current in frog olfactory receptor neurons (Pun et al., 1994).

Two types (CB₁ and CB₂) of cannabinoid receptors have been cloned from many vertebrates including mammals, birds, amphibians and fish (Lutz, 2002). CB₁ is expressed predominantly in the central and peripheral nervous system, while CB₂ is present exclusively in immune cells. CB₁ is a typical G_i/o-coupled receptor and can initiate various signaling events (Piomelli, 2003). These include closure and opening of ion channels, inhibition of adenylyl cyclase activity and stimulation of protein kinases. In the present study we demonstrate that external application of a putative endocannabinoid, 2-AG ether, and cannabinomimetic aminoalkylindole WIN 55,212-2, produced potent inhibition of the voltage-gated Na⁺ current in frog parathyroid cells, although their receptors are not found on the cells. External cannabinoids as well as internal GTPS shifted the V_1/2 for activation and inactivation, suggesting that cannabinoids could inhibit the Na⁺ current via a G protein-dependent mechanism. Although a PKC activator, PDBu, also inhibited the Na⁺ current and shifted the V_1/2 for inactivation, the modulating effect of cannabinoids on the Na⁺ current cannot be explained by PKC activity. Further studies will be required to elucidate the mechanism for modulation induced by cannabinoids.

In conventional whole-cell configuration, human parathyroid cells hardly displayed macroscopic K⁺ current in low intracellular Ca²⁺ concentration (<10 nmol l^–1; Välimäki et al., 2003). We also could not record voltage-gated outward currents in response to the depolarizing voltage steps from a holding potential of –84 mV. The free Ca²⁺ concentration of the present internal solution is calculated to be about 16 nmol l^–1 using the Webmaxc software. As expected from those results, both human and frog parathyroid cells displayed low resting potential in the condition of low intracellular Ca²⁺. In human parathyroid cells, the K⁺ current increases and the membrane potentials are hyperpolarized when extracellular Ca²⁺ concentration is elevated (Välimäki et al., 2003). On the other hand, high extracellular Ca²⁺ elicits Ca²⁺-activated Cl^– conductance in frog parathyroid cells (Okada et al., 2001). The activation of Cl^– conductance may hyperpolarize the cells if intracellular Cl^– concentration is low. Further experiments should clarify more fully the relationships between changes in extracellular Ca²⁺ concentration and the regulation of Ca²⁺-activated Cl^– channel in frog parathyroid cells.

In conclusion, frog parathyroid cells possess voltage-gated Na⁺ channels whose activity may be modulated by cannabinoids.

	List of symbols and abbreviations

TOP Summary Introduction Materials and methods Results Discussion List of symbols and... References

 

2-AG

2-arachidonoylglycerol

[Ca²⁺]_o

extracellular free Ca²⁺ concentration

1,25(OH)₂D₃

1,25-dihydroxycholecalciferol

a

maximum magnitude

CaR

Ca²⁺-sensing receptor

CT

calcitonin

DMSO

dimethyl sulphoxide

G

peak conductance

GPCR

G protein-coupled receptor

I

current

k

Boltzmann slope factor

PKA

protein kinase A

PKC

protein kinase C

PTH

parathyroid hormone

t

time

V

voltage

V_rev

apparent reversal potential

time constant

	Acknowledgments

 
This work was supported by Grants-in-Aid (14540630) from Japan Society for the Promotion of Science to Y.O.

	References

TOP Summary Introduction Materials and methods Results Discussion List of symbols and... References

 

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Bruce, B. R. and Anderson, N. C., Jr (1979). Hyperpolarization in mouse parathyroid cells by low calcium. Am. J. Physiol. 236,C15 -C21.

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