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

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⁺]_oenhances 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(Gαβγ) to Gα-GTP and Gβγ. 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.

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^–1l-cysteine and 10 units ml^–1papain (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).

Voltage-clamp recording was performed in whole-cell configuration(Hamill et al., 1981) using a CEZ 2300 patch-clamp amplifier (Nihon Kohden, Tokyo, Japan). The patch pipettes were pulled from Pyrex glass capillaries containing a fine filament(Summit Medical, Tokyo, Japan), using a two-stage puller (Narishige PD-5,Tokyo, Japan). The tips of the electrodes were heat-polished with a microforge(Narishige MF-80). The resistance of the resulting patch electrode was 5–10 MΩ when filled with internal solution. The formation 5–20 GΩ seals between the patch pipette and the cell surface was facilitated by applying weak suction to the interior of the pipette. The patch membrane was broken by applying strong suction, resulting in a sudden increase in capacitance. Amphotericin B (133–160 μg ml^–1,Sigma) was added to the pipette solution when using the perforated method(Rae et al., 1991). The perforated whole-cell condition was obtained within 5 min of the establishment of a GΩ seal. Recordings were made from parathyroid cells that had been allowed to settle on the bottom of a chamber placed on the stage of an inverted microscope (Olympus IMT-2, Tokyo, Japan). The recording pipette was positioned with a hydraulic micromanipulator (Narishige WR-88). The current signal was low-pass-filled at 5 kHz, digitized at 125 kHz using a TL-1 interface (Axon Instruments, Union City, CA, USA), acquired at a sampling rate of 2–10 kHz using a computer running the pCLAMP 5.5 software (Axon Instruments), and stored on hard disk. The pCLAMP was also used to control the digital–analogue converter for the generation of the clamp protocol. The indifferent electrode was a chlorided silver wire. The voltages were corrected for the liquid junction potential (–4 mV) between normal saline solution and the standard K⁺ internal solution. Capacitance and series resistance were compensated for, as appropriate. The series resistances after compensation in perforated mode were in the range 8–15 MΩ. The whole-cell current–voltage (I–V) relationship was obtained from the current generated by the 70 ms voltage step pulses between –74 and +56 mV in 10 mV increments from a holding potential of–84 mV. The voltage-dependence of steady-state inactivation was studied in a conventional manner by applying stepwise 200 ms conditioning pulses over the range from –124 mV to –34 mV and a single depolarization to–34 mV near the peak of the I–V curve. The time course of recovery from inactivation was measured by double-pulse protocols. Input resistance was calculated from the slope conductance generated by the voltage ramp from –104 to –54 mV.

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. GTPγS (0.5 mmol l^–1, Sigma) and GDPβS (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).

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 GΩ (13.7±0.9 GΩ, 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 GΩ (11.1±1.8 GΩ, 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.

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.

Membrane-delimited G protein-regulation of voltage-gated Na⁺channels has been suggested. We also investigated the effect of GTPγS on the Na⁺ channels in frog parathyroid cells. Activation parameters were compared with each other in the presence and absence of GTPγS. 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. GDPβS (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 GTPγS 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^–1GDPβS and –11.6±1.3 pA pF^–1 (N=8)in the condition containing 0.5 mmol l^–1 GTPγS,respectively. The voltage of half-maximum activation V_1/2in 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^–1GDPβS 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 GTPγS shifted the V_1/2 to –35.5±3.3 mV(N=8, P<0.05; Fig. 4B).

We measured inactivation time constant (τ) 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 GDPβS and 0.5 mmol l^–1 GTPγS 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 GDPβS 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 GTPγS significantly shifted the V_1/2 to –98.4±2.1 mV (N=12, P<0.05; Fig. 5C).

The time course of recovery of the inward currents from inactivation was investigated using double-pulse protocol with varying pulse intervals. The two-step pulses (control and test pulse) were identical and consisted of a depolarization to –24 mV, lasting 20 ms(Fig. 6A). The time course of recovery was single exponential (Fig. 6B). The time constant was 9.6±1.3 ms (N=5) in perforated mode and 13.1±0.7 ms (N=5) in conventional mode,respectively. Addition of 0.5 mmol l^–1 GTPγS to the internal solution further prolonged the time constant to 16.5±2.4 ms(N=5). Recovery was significantly more rapid in perforated mode than in conventional mode and the condition containing 0.5 mmol l^–1 GTPγS. However, there was no significant difference in the time constant between the conventional mode and after addition of GTPγS.

In the present study, dialysis of GTPγS into internal solution modulated activation and inactivation of Na⁺ currents, suggesting regulation by a G protein-coupled mechanism. We therefore searched for the ligand that can modulate the Na⁺ current activity. Parathyroid cells express the CaR and the PTH release from the cells is decreased by a CaR agonist, spermine (Quinn et al.,1997). Spermine (1 mmol l^-1), however, did not affect the Na⁺ current activity (Fig. 7C). By contrast, beta-adrenergic agonists stimulate cAMP production and PTH release in parathyroid cells(Brown et al., 1977). Adrenaline (10 μmol l^–1) also did not significantly inhibit the Na⁺ current (Fig. 7C).

Although the cannabinoid receptors were not found on the cell membrane of parathyroid cells, frog parathyroid cells under perforated whole-cell mode were exposed to a putative endocannabinoid, 2-AG ether (50 μmol l^–1). The drug did not change the basal properties of the cells, but reduced the Na⁺ current. The peak current at –24 mV decreased to 36±8% (P<0.05) of the controls in 5 of 5 cells (Fig. 7C). Even when the external solution was returned to normal saline solution, the Na⁺current did not recover to the initial level. A cannabinomimetic aminoalkylindole, WIN 55,212-2 (10 μmol l^–1) reversibly inhibited the Na⁺ current (Fig. 7A,B). The peak current at –24 mV decreased to 33±7%(N=5, P<0.05) of the controls. WIN 55,212-2 shifted the V_1/2 of activation by 11.7±1.6 mV (N=4, P<0.05; Fig. 8A,C)and the V_1/2 of inactivation by 17.5±1.9 mV(N=4, P<0.05; Fig. 8B,D). 2-AG ether also induced similar shift in the V_1/2 of activation and inactivation(Fig. 8C,D).

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).

We further evaluated the effect of a PKC inhibitor (chelerythrine) on the modulation of Na⁺ current elicited by cannabinoids. Chelerythrine(10 μmol l^–1) itself did not affect the Na⁺current (Fig. 11A). Even when frog parathyroid cells were pre-incubated for 10 min in a solution containing 10 μmol l^–1 chelerythrine, subsequent application of 10μmol l^–1 WIN 55,212-2 still inhibited the Na⁺currents (Fig. 11A,B). Furthermore, in the presence of PKC inhibitor, WIN 55,212-2 shifted the V_1/2 of activation by 11.3±2.6 mV (N=3, P<0.05; Fig. 11C)and the V_1/2 of inactivation by 18.3±5.0 mV(N=3, P<0.05; Fig. 11D). These results indicate that WIN 55,212-2 acts on the Na⁺ currents, probably through a mechanism independent of PKC.

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 GTPγS, 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 GTPγS, the mean V_1/2 for Na⁺ current activation was about –46 mV. A similar mean value was observed in the presence of GDPβS in the pipette solution. Since internal GTPγS shifted the V_1/2 to a more positive level (–36 mV),the presence of GTPγS results in the activation of a G protein-dependent mechanism, which may be functionally important in regulating Na⁺current activation. A similar effect of GTPγS 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 GTPγS inhibits the Na⁺ channel by shifting the steady-state inactivation to more negative potentials (Schubert et al., 1989 and 1990). In contrast,GTPγS 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 GTPγS 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.

 
• 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

 
• τ

  time constant

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