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Logo of jphysiolThe Journal of Physiology SiteMembershipSubmissionJ Physiol
J Physiol. Feb 15, 1998; 507(Pt 1): 71–75.
PMCID: PMC2230766

Endomorphins inhibit high-threshold Ca2+ channel currents in rodent NG108-15 cells overexpressing μ-opioid receptors

Abstract

  1. Extracellular application of the novel brain peptides endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited high-threshold Ca2+ channel currents in NGMO-251 cells, a daughter clone of NG108-15 mouse neuroblastoma × rat glioma hybrid cells, in which μ-opioid receptors are overexpressed.
  2. In contrast, EM1 and EM2 did not induce this inhibition in the parental NG108-15 cells that predominantly express endogenous δ-receptors.
  3. The IC50 for EM1 and EM2 was 7.7 and 23.1 nm, respectively.
  4. EM-induced Ca2+ channel current inhibition was blocked by treatment or pretreatment of the cells with 100 μmN-methylmaleimide or 100 ng ml−1 pertussis toxin.
  5. These results show that a decrease in conductance of Ca2+ channels results following interaction of EMs with cloned μ-receptors, which couple via Gi/Go-type G proteins, and that EMs fulfill one of the necessary synaptic conditions for them to be identified as neurotransmitters.

Endomorphin 1 (EM1) and endomorphin 2 (EM2), recently isolated from bovine brain, are tetrapeptides with C-terminal amidation (Zadina, Hackler, Ge & Kastin, 1997). EMs have been shown to have the highest affinity and specificity for μ-opioid receptors by ligand-binding assay (Zadina et al. 1997). EM-like immunoreactivity is found in thalamus, hypothalamus, striatum and frontal cortex (Zadina et al. 1997), where μ-opioid receptors are concentrated (George et al. 1994; Bunzow et al. 1995). The question arises whether EMs function as neurotransmitters to mediate analgesia, opioid dependence and neuroendocrine effects.

If we assume that EMs are neurotransmitters, EMs should fulfill several criteria. A crucial criterion is that EMs should activate or inhibit G proteins and effector enzymes to produce second messengers, or alternatively, EMs should change ionic conductance after interacting with μ-receptors in the postsynaptic or presynaptic membranes.

To pursue the above hypothesis, we used cultured NGMO-251 cells that overexpress cloned rat μ-opioid receptors (Morikawa, Fukuda, Kato, Mori & Higashida, 1995). The parental NG108-15 cells were transfected with cDNA encoding the μ-receptor (Fukuda, Kato, Mori, Nishi & Takeshima, 1993) so that NGMO-251 cells represent a transformant of NG108-15 mouse neuroblastoma × rat glioma hybrid cells (Nirenberg et al. 1983). NGMO-251 cells maintain low- and high-threshold Ca2+ currents (Morikawa et al. 1995), as described in the parental NG108-15 cells (Tsunoo, Yoshii & Narahashi, 1986; Hescheler, Rosenthal, Trautwein & Schultz, 1987; Brown, Docherty & McFadzean, 1989; Kasai & Neher, 1992). ω-Conotoxin-sensitive (N-type) Ca2+ channel currents of NGMO-251 cells containing both μ- and δ-receptors are inhibited by the μ-agonist [D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and the δ-agonist [D-Pen2,D-Pen5]-enkephalin (DPDPE), whereas the parental NG108-15 cells are only inhibited by DPDPE (Morikawa et al. 1995). Therefore, NGMO-251 cells are a good neuronal model for investigating agonist-induced conductance changes in ion channels. Here we demonstrate that EMs induce an inhibition of high-threshold Ca2+ channel currents in the μ-containing NGMO-251 cells, but not in the δ-containing NG108-15 cells (Evans, Keith, Morrison, Magendzo & Edwards, 1992), in a N-methylmaleimide (NMM)- and pertussis toxin (PTX)-sensitive manner.

METHODS

Cell lines

The expression plasmid pRORS15-1 (Fukuda et al. 1993) was constructed by inserting the entire protein coding region of the rat μ-opioid receptor cDNA into the vector pKNH, which carries the SV40 early gene promotor and the neomycin-resistant marker gene (Morikawa et al. 1995). NG108-15 cells, obtained from Dr Nirenberg's laboratory, National Institutes of Health, USA, were transfected with pRORS15-1 by the calcium phosphate method as described by Morikawa et al. (1995). Neomycin-resistant clones expressing the μ-opioid receptor were isolated by screening clones with RNA blot hybridization analysis using the rat μ-opioid receptor cDNA as a probe. The NGMO-251 cell line is one of two established clones (Morikawa et al. 1995).

Cell culture

NGMO-251 and NG108-15 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco Life Technologies) supplemented with 5 % fetal calf serum, 100 μM hypoxanthine, 1 μM aminopterin and 16 μM thymidine, at 37°C in a humidified atmosphere of 90 % air and 10 % CO2 (Morikawa et al. 1995). For electrophysiological measurements, cells were plated onto 35 mm diameter plastic dishes coated with 0.01 % poly-ornithine (Higashida, Hashii, Fukuda, Caulfield, Numa & Brown, 1990). The cells were further cultured for differentiation for 10-14 days in DMEM supplemented with 1 % fetal calf serum, 100 μM hypoxanthine, 16 μM thymidine and 0.25 mM dibutyryl cyclic AMP, as described previously (Higashida et al. 1990; Kasai & Neher, 1992).

Electrophysiological recordings

Ca2+ channel currents were measured by the whole-cell clamp method with an Axoclamp-2A patch-clamp amplifier (Axon Instruments), as described previously (Brown et al. 1989). The recording chamber was superfused with Ba2+-containing solution consisting of 50 mM BaCl2, 30 mM NaCl, 5 mM CsCl, 25 mM tetraethylammonium chloride, 25 mM glucose, 0.1 μM tetrodotoxin and 5 mM sodium Hepes, pH 7.2 (Tsunoo et al. 1986; Higashida et al. 1990). Patch pipettes were filled with a Cs+-rich solution containing 150 mM CsCl, 1 mM MgCl2, 1.1 mM NaEGTA, 0.4 mM Na2ATP and 10 mM caesium Hepes, pH 7.2 (Higashida et al. 1990). Pipette resistances ranged between 4 and 8 MΩ. Currents were low-pass filtered (0.3-1 kHz), sampled at 1 kHz, and analysed by pCLAMP (Axon Instruments) with a digital computer. To eliminate capacitative and leakage currents, a P/4 procedure was used (Bezanilla & Armstrong, 1977). The membrane potential was held at -80 mV and step depolarized for 200 ms to -20 and +20 mV every 30 s to generate low- and high-threshold voltage-activated Ca2+ channel currents (Nomura, Reuveny & Narahashi, 1994).

Current amplitude was measured at the peak (10-20 ms after the onset of depolarization) under control conditions. NGMO-251 cells generated low voltage-activated (LVA) transient Ca2+ currents (‘T-type’ currents) of < 2 nA at -20 mV. We used cells with small LVA currents to minimize contamination by the T-type current.

All values are represented as means ±s.e.m. Homogeneity of variances was tested with Fisher's F test followed by Student's t test (homogeneous variances) or Welch's t test (non-homogeneous variances) to compare the effects of two experimental conditions for parallel groups. Statistical significance was accepted when P < 0.01.

Endomorphins

EMs used in the present experiments were isolated from bovine frontal cortex by the method described by Zadina et al. (1997). DPDPE was purchased from Peptide Institute Inc. (Osaka, Japan).

RESULTS

EM1 and EM2 reversibly reduced the peak amplitude of high-threshold Ca2+ channel currents (ICa(h)) without any effect on the transient LVA current measured with Ba2+ as a charge carrier in NGMO-251 cells. Figure 1 illustrates examples of double-pulse protocol experiments. The transient LVA Ca2+ channel current (associated with the pulse to -20 mV) was not changed by application of 100 nM EM1 or EM2 through ejection pipettes onto the surface of the medium just above the cells (control and test currents overlap; Fig. 1A and B). ICa(h) associated with the pulse to +20 mV was reduced and recovered to the original level. EM1 and EM2 preferentially suppressed the transient component rather than the long-lasting component and also markedly slowed current onset (data not shown). This was frequently encountered with higher agonist concentrations. The EM inhibition was antagonized by 10 μM naloxone (Fig. 1D and E).

Figure 1
Effect of EM1, EM2 and DPDPE on low- and high-threshold Ca2+ channel currents in NGMO-251 cells

The mean inhibition of ICa(h) by 100 nM EM1 and EM2 by the focal application method was 29.1 ± 1.97 % (n= 8) and 21.8 ± 2.99 % (n= 13), respectively; these values were significantly different from those prior to EM application (P < 0.001; Table 1). This large inhibition was comparable to the results with 1 μM DAMGO in NGMO-251 and NGMO-225 cells transformed to express μ-receptors, but not in the parental NG108-15 cells expressing predominantly δ-receptors (Morikawa et al. 1995). A current-voltage relationship was observed in the presence and absence of 10 nM EM1 (Fig. 2A) and EM2 (Fig. 2B). Maximum inhibition was obtained with depolarizing steps to +20 mV.

Figure 2
EM1- and EM2-induced depression of ICa(h) recorded from an NGMO-251 cell
Table 1
Inhibition of ICa(h) by EM1, EM2 and DPDPE in NG108–15 cells and transformed NG108–15 cells (NGMO-251) expressing μ-opioid receptors

The inhibition of ICa(h) by EM was concentration dependent (Fig. 3). The IC50 values of 7.7 nM for EM1 and 23.1 nM for EM2 were obtained by bath-perfusion experiments. These values are about 10-fold higher than Ki values (0.36 nM for EM1 and 0.64 nM for EM2) estimated from receptor-binding assays (Zadina et al. 1997).

Figure 3
Dose-response relationship for inhibition of ICa(h) by EM1 and EM2

The inhibition of ICa(h) by EM was completely reversed by both treatment or pretreatment with 100 μM NMM (Table 1), an analogue of N-ethylmaleimide which is known to be a sulfhydryl alkylating agent that inhibits G protein action in NG108-15 cells (Kasai, 1991) and rat superior cervical ganglion neurons (Shapiro, Wollmuth & Hille, 1994). Figure 1G and H depicts examples of an experiment in which 100 nM EM1 and EM2 had no effect on the amplitude of ICa(h) after 100 μM NMM. The effect of NMM was not reversible after washout, since repeated applications of EM1 and EM2 failed to cause inhibition of current. The same pharmacological effects on inhibition of ICa(h) by DPDPE in NG108-15 cells were observed following pretreatment with NMM (Table 1).

Pretreatment with 100 ng ml−1 PTX for 18 h also completely eliminated the inhibition by EM1 and EM2 (Fig. 1I and J, and Table 1). The effect of PTX on μ-receptors in NGMO-251 cells, which mediate inhibition of ICa(h) by DAMGO, has been reported previously (Morikawa et al. 1995). These results indicate that EM inhibition is mediated by the activation of G proteins (Gi/Go).

Little or no inhibition of ICa(h) was induced by focal application of 100 nM EM1 and EM2 to the parental NG108-15 cells (Table 1), which lack μ-receptors. In contrast, substantial inhibition of 30.7 ± 4.90 % (n= 7; P < 0.001) and 21.2 ± 1.91 % (n= 5; P < 0.001) was obtained by application of 100 nM DPDPE to NG108-15 cells (current traces not shown) and NGMO-251 cells (Fig. 1C and Table 1), reflecting the response mediated by endogenous δ-receptors known to be present in both cell lines. The δ-receptor-mediated inhibition by DPDPE was also blocked by 10 μM naloxone (Fig. 1F).

Repetitive application of homologous (same peptide) or heterologous EM peptide reduced or abolished subsequent inhibition by EM. This cross-desensitization suggests that EM1 and EM2 act on the same μ-receptor sites.

DISCUSSION

The results clearly show that the brain neuropeptides EM1 and EM2 resulted in marked inhibition of the high voltage-activated Ca2+ channel current after interacting with cloned μ-receptors expressed in NGMO-251 cells and that the action of EMs on Ca2+ channels was inhibited by the opiate receptor antagonist, naloxone. This is in agreement with the idea that opioids inhibit Ca2+ channel currents in various neuronal preparations. Similar inhibition has been shown in NG108-15 cells with noradrenaline (Brown et al. 1989) and acetylcholine (Higashida et al. 1990).

Ca2+ current inhibited by EM1 and EM2 in NGMO-251 cells is mostly N-type current, since it has been previously shown that the ω-conotoxin-sensitive current is inhibited to the same extent by DAMGO in μ-receptor-overexpressing NGMO-251 cells and by DPDPE in parental NG108-15 cells (Morikawa et al. 1995). Additional pharmacological experiments are necessary to determine whether or not P- and Q-type Ca2+ currents are inhibited by EMs. Our results are consistent with previous observations showing that the N-type (high-threshold) voltage-dependent Ca2+ current is inhibited by activation of pharmacologically defined μ-receptors in rat dorsal ganglion neurons (Moises, Rusin & Macdonald, 1994; Nomura et al. 1994). More recently, α1B (N-type) Ca2+ channels have been shown to be inhibited by stimulation of cloned μ-opioid receptors co-expressed in Xenopus oocytes (Bourinet, Soong, Stea & Snutch, 1996).

The inhibition by EM was abolished by both NMM and PTX, suggesting that the effects of the EMs are mediated by Gi/Go-type G proteins. This agrees well with the recent observation that coupling between μ-opioid receptors and N-type Ca2+ channels is mediated either by α- or βγ-subunits of G proteins (Bourinet et al. 1996).

In conclusion, our results in NG108-15 cells indicate that the novel peptides EM1 and EM2 function to modulate conductance of voltage-activated Ca2+ channels.

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