Modulation of High-Voltage Activated Ca2+ Channels in the Rat Periaqueductal Gray Neurons by µ-Type Opioid Agonist

Chang-Ju Kim, Jeong-Seop Rhee, and Norio Akaike

Department of Physiology, Kyung Hee University College of Medicine, Seoul, Korea; and Department of Physiology, Kyushu University Faculty of Medicine, Fukuoka 812-82, Japan

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Kim, Chang-Ju, Jeong-Seop Rhee, and Norio Akaike. Modulation of high-voltage activated Ca2+ channels in the rat periaqueductal gray neurons by µ-type opioid agonist. J. Neurophysiol. 77: 1418-1424, 1997. The effect of µ-type opioid receptor agonist, D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO), on high-voltage-activated (HVA) Ca2+ channels in the dissociated rat periaqueductal gray (PAG) neurons was investigated by the use of nystatin-perforated patch recording mode under voltage-clamp condition. Among 118 PAG neurons tested, the HVA Ca2+ channels of 38 neurons (32%) were inhibited by DAMGO (DAMGO-sensitive cells), and the other 80 neurons (68%) were not affected by DAMGO (DAMGO-insensitive cells). The N-, P-, L-, Q-, and R-type Ca2+ channel components in DAMGO-insensitive cells shared 26.9, 37.1, 22.3, 7.9, and 5.8%, respectively, of the total Ca2+ channel current. The channel components of DAMGO-sensitive cells were 45.6, 25.7, 21.7, 4.6, and 2.4%, respectively. The HVA Ca2+ current of DAMGO-sensitive neurons was inhibited by DAMGO in a concentration-, time-, and voltage-dependent manner. Application of omega -conotoxin-GVIA occluded the inhibitory effect of DAMGO ~70%. So, HVA Ca2+ channels inhibited by DAMGO were mainly the N-type Ca2+ channels. The inhibitory effect of DAMGO on HVA Ca2+ channels was prevented almost completely by the pretreatment of pertussis toxin (PTX) for 8-10 h, suggesting that DAMGO modulation on N-type Ca2+ channels in rat PAG neurons is mediated by PTX-sensitive G proteins. These results indicate that µ-type opioid receptor modulates N-type HVA Ca2+ channels via PTX-sensitive G proteins in PAG neurons of rats.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Opioid peptides and opiates produce analgesia by activating the descending pain modulatory pathways (Duggan et al. 1983; Fields et al. 1991; Light et al. 1986; Renno et al. 1992) and exert their effects by interacting with receptors, referred to as µ, delta , and kappa  (Crain and Shen 1990; Reisine and Bell 1993). The µ-receptors are involved predominantly in the periaqueductal gray (PAG) region (Mansour et al. 1995). Focal electric stimulation in the PAG matter and microinjection of morphine into PAG produces a profound antinociception, and this effect also was antagonized by naloxone (Akil et al. 1976; Yaksh et al. 1976). Therefore endogenous opioids have been postulated to play an important role in analgesia especially at the level of the midbrain PAG. The major effect of enkephalin on PAG cells was reported as inhibition probably due to the inhibitory cellular action (Behbehani et al. 1990).

It has been demonstrated that several different types of Ca2+ channels exist in neurons. From the functional point of view, the Ca2+ channels have been classed into "high" and "low" threshold on the basis of the voltage range at which they are activated. Low-voltage-activated Ca2+ channels are known as T-type Ca2+ channels (Akaike et al. 1989; Takahashi and Akaike 1991). High-voltage activated (HVA) Ca2+ channels could be classified pharmacologically into five types: dihydropyridine (DHP)-sensitive L-typechannel; omega -conotoxin-GVIA-sensitive N-type channel;omega -agatoxin-IVA-sensitive P-type channel; omega -conotoxinMVIIC-sensitive but DHP/omega -conotoxin-GVIA/omega -agatoxin-IVA-insensitive Q-type channel; and resistant R-type channel (Dunlap et al. 1995; Nowycky et al. 1985; Zhang et al. 1993).

It has been suggested that opiates regulate the nociceptive transmission in part by inhibiting the release of transmitters (Duggan 1983; Jessell and Iversen 1977; Yaksh et al. 1980). The effect of opiates and opioid peptides also have been reported due to the activation of K+ channels (Madison and Nicoll 1988; Yoshimura and North 1983) or inhibition of Ca2+ channels (Schroeder et al. 1991; Toth et al. 1993). There is evidence that Ca2+ channels primarily regulate the transmitter release from peripheral (Hille 1994; Toth et al. 1993) and central neurons (Takahashi and Momiyama 1993). However, the regulation of Ca2+ channels by opioid receptors in PAG neurons remained to be clarified. In this study, we investigated the modulation of HVA Ca2+ channels in the acutely dissociated rat PAG neurons by use of nystatin-perforated patch-clamp technique and tried to discover which types of HVA Ca2+ channels are modulated by µ-type opioid receptor.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Cell preparation

PAG neurons were dissociated using a technique described elsewhere (Kaneda et al. 1988). Briefly, 10- to 16-day-old Wistar rats of both sexes were decapitated under pentobarbital sodium anesthesia. The brain was removed and 400-µm-thick slices containing PAG neurons were obtained by using a microslicer (DSK, model DTK-1000, Japan). The brain slices were pre-incubated in an incubation solution well saturated with 95% O2-5% CO2 at room temperature for 30 min. Thereafter, the slices were treated with 1 mg/6 ml pronase for 20-30 min at 32°C and subsequently exposed to 1 mg/6 ml thermolysin under the same conditions. After enzyme treatment, the slices were kept in an enzyme-free incubation solution. The portion of PAG neurons was removed by micropunching and mechanically dissociated with fire-polished Pasteur micropipettes in a small plastic culture dish (diameter 35 mm; Falcon) filled with standard solution. The dissociated neurons adhered to the bottom of the dish within 20 min. However, the experiments using pertussis toxin (PTX) were done on dissociated neurons pretreated with or without 500 ng/ml PTX for 8-10 h at room temperature (20-22°C).

Solutions

The ionic composition of the incubation solution was (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 10 glucose, and 24 NaHCO3 and was bubbled continuously by 95% O2-5% CO2. The standard solution used was (in mM) 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The pH was adjusted to 7.4 with tris (hydroxymethyl) aminomethane (Tris-base). The external solution for the recording of Ba2+ current passing through the voltage-gated Ca2+ channels was (in mM) 145 NaCl, 5 CsCl, 5 BaCl2, 1 MgCl2, 10 glucose, and 10 HEPES. This external Ba2+ solution contained 3 × 10-7 M tetrodotoxin (TTX), at which concentration the voltage-dependent Na+ channel was blocked completely. The patch-pipette solution for the nystatin-perforated patch recording mode contained (in mM) 150 CsCl and 10 HEPES. The pH was adjusted to 7.2 by adding Tris-base. A stock solution containing 10 mg/ml nystatin was prepared and added in a final concentration of 200 µg/ml to the patch-pipette solution.

Drugs

Drugs used in these experiments were: pronase (Calbiochem, La Jolla, CA), thermolysin, nystatin, nicardipine, pertussis toxin (PTX), D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO), dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO), omega -conotoxin-GVIA, omega -agatoxin-IVA, omega -conotoxin-MVIIC (Peptide Institute, Osaka, Japan), and TTX (Sankyo, Tokyo, Japan). Experiment using nicardipine was carried out in the dark room. Nicardipine was dissolved in DMSO and further diluted in external solution. Other drugs were dissolved directly in the external solution before use.

Drugs were applied using a rapid application system termed the "Y-tube method" as described elsewhere (Murase et al. 1990). By this technique, the external solution surrounding a neuron could be exchanged within 10-20 ms.

Electrical measurements

Electrical recordings were performed using the nystatin-perforated patch recording configuration. Patch pipette was prepared from glass capillaries with an outer diameter of 1.5 mm on a two-stage puller (PB-7, Narishige, Tokyo, Japan). The resistance between the recording electrode filled with the internal solution and the reference electrode was 6-9 MOmega . After stable perforated patch formation, the series resistance ranged from 16 to 25 MOmega . Ionic currents and voltages were measured with a patch clamp amplifier (EPC-7, List-Electronic, Darmstadt/Eberstadt, Germany), monitored on a storage oscilloscope (HS-5100A, Iwatsu, Tokyo, Japan, and simultaneously recorded on video tapes after digitization with a PCM processor (PCM 501 ESN, Nihon Kohden, Tokyo, Japan).

The data was analyzed by an IBM PC 386 using the pCLAMP software. Leak sweeps were obtained at several intervals during the experiment by averaging 10 hyperpolarizing test pulses. Leak current was subtracted from the data sweeps by scaling the leak sweep to the data. All experiments were carried out at room temperature (20-22°C).

Statistical analysis

Experimental values are presented as means ± SE. Results were analyzed by Student's t-test.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Acutely dissociated PAG neurons

In the use of nystatin-perforated patch recording mode, HVA Ba2+ current (IBa) passing through the HVA Ca2+ channels was recorded from all 118 PAG neurons tested under the experimental conditions that suppress the Na+ and K+ channels. This perforated patch technique prevented the run-down of HVA IBa and stabilized current recordings (Ye and Akaike 1993). The application of 10-6 M DAMGO inhibited the HVA IBa elicited by depolarizing step pulse from a holding potential of -50 to 0 mV in 38 (32%) PAG neurons. In the other 80 neurons, DAMGO had no significant effects. Thus with respect to Ca2+ channel inhibition, the neurons were classified conventionally into two classes, DAMGO-sensitive and -insensitive cells. Generally, DAMGO-sensitive cells had small- or medium-sized somas and thin dendrites (Fig. 1), whereas DAMGO-insensitive cells had large somas and thick dendrites.


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FIG. 1. Morphology of acutely isolated periaqueductal gray (PAG) neurons. This sample of neuron was dissociated freshly from 12-day-old rat. Scale bar indicates 10 µm.

Inhibition of HVA Ca2+ channels by DAMGO

In the present study, the modulation of HVA Ca2+ channels was examined in DAMGO-sensitive neurons. As shown in Fig. 2A, the HVA IBa evoked by the depolarizing step from VH of -50 to 0 mV had a rapid rising phase and decayed little during the 300-ms depolarizing step pulse. The application of DAMGO inhibited HVA IBa in which the inhibitory effect was eliminated gradually during a continuous depolarizing step pulse. In the present experiments, the current amplitudes were measured arbitrarily at 10 ms after the onset of a depolarizing step pulse in the presence and absence of DAMGO, and the results are shown by percent inhibition in comparison with each other.


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FIG. 2. Inhibition of high-voltage-activated (HVA) IBa by D-Ala2,N-MePhe4,Gly5-ol-enkephalin (DAMGO) in DAMGO-sensitive neurons. A: DAMGO-induced inhibition of HVA IBa elicited by a 300-ms depolarizing step from VH of -50 to 0 mV. B: representive current-voltage (I-V) relationships of HVA IBa in absence (open circle ) and presence (bullet ) of 10-6 M DAMGO. C: concentration-inhibition curve of HVA IBa evoked by a 30-ms depolarizing step from VH of -50 to 0 mV in the presence of DAMGO of various concentrations.

Figure 2B illustrates the current-voltage (I-V) relationship of HVA IBa in the absence and presence of 10-6 M DAMGO. The inhibitory effect of DAMGO was strongly eliminated at membrane potentials more positive than +20 mV. When the concentration-inhibition curve for DAMGO-induced inhibition was made on HVA IBa elicited by a depolarizing step pulse from VH of -50 to 0 mV, the inhibitory effect was saturated at high concentrations beyond 10-5 M. The maximum inhibition induced by 3 × 10-5 M DAMGO was 22.2 ± 5.1% (n = 5), and the half-maximal inhibitory concentration (IC50) was 6.5 × 10-7 M (Fig. 2C).

HVA Ca2+ channel subtypes in DAMGO-sensitive and -insensitive neurons

In the present study, HVA Ca2+ channel current in DAMGO-sensitive and -insensitive neurons was fractionated into N, P, L, Q, and R type using selective Ca2+ antagonists. N-, P-, L-, and Q-type Ca2+ channel current was defined as the current blocked by 3 × 10-6 M omega -conotoxin-GVIA,3 × 10-7 M omega -agatoxin-IVA, 5 × 10-6 M nicardipine, and3 × 10-6 M omega -conotoxin-MVIIC, respectively. As shown in Fig. 3A, the successive reduction of the HVA IBa was observed using cumulative application of these blockers at respective saturation concentrations. A small but measurable fraction of R-type current components remains in the presence of four organic antagonists.


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FIG. 3. Pharmacological separation of HVA IBa. A: representive currents of HVA IBa in DAMGO-insensitive and -sensitive neurons. Ca2+ channel antagonists were applied with cumulative addition of 3 × 10-6 M omega -conotoxin-GVIA, 3 × 10-7 omega -agatoxin-IVA, 5 × 10-6 M nicardipine, 3 × 10-6 M omega -conotoxin-MVIIC, and 10-4 M Cd2+. Currents were elicited by a 30-ms depolarization step pulse from VH of -50 to 0 mV at 20-s intervals. B: difference of HVA Ca2+ channel components in DAMGO-insensitive and -sensitive neurons. All data was obtained from 5 neurons.Asterisk shows a significant difference at P < 0.05.

In DAMGO-insensitive neurons, N-, P-, L-, Q-, and R-type current components shared 26.9 ± 4.2%, 37.1 ± 9.5%, 22.3 ± 7.5%, 7.9 ± 3.1%, and 5.8 ± 3.1%, respectively. In the case of DAMGO-sensitive cells, N-, P-, L-, Q-, and R-type current components were 45.6 ± 4.4, 25.7 ± 3.8,21.7 ± 4.9, 4.6 ± 0.8, and 2.4 ± 1.6% of the total control current, respectively (n = 5; Fig. 3); this indicated that DAMGO-sensitive cells have 1.7 times larger N-type channel components than do DAMGO-insensitive cells.

Types of Ca2+ channels inhibited by DAMGO

To know which subtypes of HVA Ca2+ channels in DAMGO-sensitive PAG neurons are inhibited by the activation of type of opiod receptors, we tested the action of DAMGO on the respective HVA Ca2+ channel components of PAG neurons in the presence of these Ca2+ channel blockers. The suppression of HVA IBa by 10-6 M DAMGO was measured before and after the cumulative application of omega -conotoxin-GVIA, omega -agatoxin-IVA, nicardipine, omega -conotoxin-MVIIC, and Cd2+ (Fig. 4). Consequently, total inhibition of HVA Ca2+ channels by DAMGO were shared70.2 ± 5.2% in N-type, 15.7 ± 8.2% in P-type, 7.2 ± 4.0% in L-type, 6.9 ± 3.1% in Q-type channel, and no effect in R-type HVA Ca2+ channels (n = 5).


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FIG. 4. Inhibitory effects of DAMGO on HVA IBa in DAMGO-sensitive neurons. A: representive currents of HVA IBa in absence (open circle ) and presence (bullet ) of 10-6 M DAMGO; 3 × 10-6 M omega -conotoxin-GVIA, 3 × 10-7 omega -agatoxin-IVA, 5 × 10-6 M nicardipine, 3 × 10-6 M omega -conotoxin-MVIIC, and 10-4 M Cd2+ were added cumulatively. Currents were elicited by a 30-ms depolarization step pulse from VH of -50 to 0 mV at 20-s intervals. B: inhibition of each Ca2+ channel component by DAMGO. All data was obtained from 5 neurons. Two asterisks show a significant suppression at P < 0.01.

To show that the order of antagonist addition did not affect the conclusion, we changed the order of antagonist addition in DAMGO-sensitive cells, which were omega -agatoxin-IVA, omega -conotoxin-GVIA, nicardipine, and omega -conotoxin-MVIIC. N-, P-, L-, Q-, and R-type current components were 53.2 ± 3.7, 20.3 ± 4.9, 18.3 ± 4.8, 4.4 ± 3.2, and 3.8 ± 0.9 of the total current, respectively (n = 5). And total inhibition of HVA Ca2+ channels by DAMGO was 68.0 ± 9.2 in N-type, 9.9 ± 3.3 in P-type, 10.1 ± 4.0 in L-type, 8.1 ± 4.1 in Q-type, and no effect in R-type HVA Ca2+ channels (n = 5).

The results suggest that DAMGO selectively inhibited mainly the N-type HVA Ca2+ channel in DAMGO-sensitive PAG neurons.

Involvement of PTX sensitive G-proteins

Opioid actions on Ca2+ channels are known to be mediated by PTX-sensitive G proteins (Rhim and Miller 1994; Taussig et al. 1992). To evaluate the involvement of PTX-sensitive G proteins on DAMGO-induced inhibition of HVA IBa in PAG neurons, the neuron was immersed in an external solution without PTX for 8-10 h. With the use of nystatin-perforated patch recording mode, the inhibition of HVA IBa, induced by 10-6 M DAMGO in control neurons that were soaked in the external solution without PTX for 8-10 h, was 23.7 ± 2.3% (n = 5). Next, the dissociated PAG neurons were incubated for 8-10 h in the external solution with PTX at a concentration of 500 ng/ml. In PTX-treated neurons, 10-6 M DAMGO inhibited HVA IBa only by4.5 ± 1.6% (n = 5; Fig. 5C). PTX, which inactivates Gi- and Go-type G proteins (Aghajanian and Wang 1986; Wimpey and Chavkin 1991), almost totally abolished DAMGO-induced inhibition of HVA IBa (Fig. 5B). The result suggests that G proteins are involved in the coupling between µ-type opioid receptors and HVA Ca2+ channels in PAG neurons.


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FIG. 5. Effects of pertussis toxin (PTX) on DAMGO-induced inhibition of HVA IBa. A: HVA IBa recorded from PAG neuron in absence and presence of 10-6 M DAMGO. Neuron was immersed in an external solution without PTX for 8-10 h. B: effect of 10-6 M DAMGO on HVA IBa in a neuron soaked in external solution containing 500 ng/ml PTX for 8-10 h. C: effects of DAMGO on HVA IBa of control (n = 5), PTX-treated (n = 5) neurons for 8-10 h. Two asterisks show significant difference from control at P < 0.01.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The present study indicated the existence of L-, N-, P-, Q-, and R-type HVA Ca2+ channels in acutely dissociated PAG neurons. The distribution ratio of these five types of channel components was different between DAMGO-insensitive and -sensitive cells. It also was noted that the DAMGO-sensitive neurons have ~1.7 times larger N-type Ca2+ channel components than DAMGO-insensitive neurons. HVA Ca2+ channels are oligometric complex of four different subunits; alpha 1, alpha 2/delta 1, beta , and gamma . And at least six different genes encoding alpha 1 subunits and three distinct genes encoding subunits have been isolated from various tissues (Vardi et al. 1991; Zhang et al. 1993). The alpha 1 subunit alone is sufficient to direct the permeation of Ca2+ and has specific receptor sites for Ca2+ channel antagonists. Thus the different distribution of the HVA Ca2+ channel subtype between DAMGO-sensitive and -insensitive neurons may result from the different expression of alpha 1 subunits in PAG neurons.

It has been reported that in the nucleus tractus solitarius of the rat, the activation of µ-receptor by DAMGO inhibits N- and P-type Ca2+ channels (Rhim and Miller 1994). In the present study, however, the µ-type opioid receptor agonist mainly inhibited N-type Ca2+ channels, but has no significant effect on P-type Ca2+ channels in PAG neurons. Therefore, µ-type opioid receptors may regulate HVA Ca2+ channels differentially in different brain regions. The present results are consistent with the selective inhibition of N-type Ca2+ channels by opioid peptides in peripheral neurons (Shen and Suprenant 1991). The activation of µ-type opioid receptors inhibits neuronal adenylate cyclase activity (Beitner et al. 1989; Reisine and Bell 1993) and increases membrane K+ conductance in a variety of central and peripheral neurons (Loose and Kelley 1990; Wimpey and Chavkin 1991). Both of these effects are blocked by PTX, suggesting that they are mediated by Gi- or Go-type G proteins (Aghajanian and Wang 1986; Wimpey and Chavkin 1991).

In vertebrate neurons, many neurotransmitters modulate Ca2+ channels via PTX-sensitive G proteins (Hescheler et al. 1987; Scholz and Miller 1991; Surprenant et al. 1990; Tatsumi et al. 1990). The present results also show that the PTX-sensitive G protein was involved in the µ-type opioid receptor-mediated inhibition of HVA Ca2+ channels in PAG neurons. A previous study using antisense oligonucleotides complementary to sequence of Go indicates that µ-type opioid receptor is coupled to Go-type of G-proteins, which induce inhibition of Ca2+ channels in rat DRG neurons (Moises et al. 1994). In this regard, the identification of a specific G protein involved in the DAMGO effect in PAG neurons will require further experiments with antisense oligonucleotide.

The present results were obtained from neuronal soma of PAG neurons with proximal dendrites. The inhibition of Ca2+ influx into the cell body by DAMGO may have a variety of effects including the regulation of Ca2+-dependent ion channels, gene expression, and metabolism (Rhim and Miller 1994). Systemic opiates caused a significant decrease in gamma -aminobutyric acid (GABA) release in the midbrain PAG (Renno et al. 1992) and the activation of µ-type opioid receptor inhibited GABA release presynaptically by G-protein-mediated inhibition (Capogna et al. 1993). The N-type Ca2+ channels play an important role in the regulation of the release of neurotransmitters from the nerve terminals (Lovinger et al. 1994; Sheng et al. 1994; Toth et al. 1993). If the N-type Ca2+ channels in the nerve terminals are coupled with µ-type opioid receptors through PTX-sensitive G proteins, similar to those found in the neuronal soma, the inhibition of the N-type Ca2+ channels by opioid would reduce the release of neurotransmitters in the PAG matter.

The PAG region of the brain is known to be involved heavily with nociception. In the PAG region, however, DAMGO regulation of HVA Ca2+ channels has not been reported previously. Thus our results suggest that modulation of N-type Ca2+ channels by µ-type opioid agonist is an important mechanism of descending pain control system in the PAG region.

    ACKNOWLEDGEMENTS

  This work was supported by the Kyung Hee University Research Fund to C.-J. Kim and by Grants-in-Aid for Scientific Research (04304028, 04404023, and 05271202) to N. Akaike from The Ministry of Education, Science and Culture, Japan, and by the Research Fund of Korea Science and Engineering Foundation and Japan Society for the Promotion of Science.

    FOOTNOTES

  Address for reprint requests: C.-J. Kim, Dept. of Physiology, Kyung Hee University College of Medicine, Tongdaemoon-Gu, Seoul, 130-701, Korea.

  Received 22 August 1995; accepted in final form 19 September 1996.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society