Involvement Of G Proteins And Ion Channels In Spinal Delta Opioid Receptormediated Antinociception

Molecular analysis of the opioid receptors indicates that they conform to the structural motif of the G protein receptor family. All three types of opioid receptors contain cysteine residues believed to be involved in disulfide bonds, and a cysteine fatty acid attachment site in locations common with other G protein-coupled receptors. All three opioid receptors contain consensus asparagine-linked glycosylation sites in the extracellular N-terminal domain as well as many consensus protein kinase sites in the first and third intra-cellular loops, and in the C-terminal domain.

Receptors that interact with G proteins produce an increase in GTP hydrolysis, ultimately via an increase in the GTPase activity of Ga, but initially by stimulating the binding of GTP to Ga [81]. Thus, a study of the ability of various receptor agonists to stimulate GTPase activity is useful to determine the nature of the interaction between the G protein and receptor.

The high-affinity GTPase activity in the mouse spinal cord is increased in a concentration-dependent manner by [D-Ala2]deltorphin II [82]. This increase of GTPase activity induced by [D-Ala2]deltorphin II is completely blocked by coincubation with a selective delta opioid receptor antagonist NTB [82].

The activation of G proteins by the opioid receptor agonist can be also measured by assessing agonist-induced stimulation of membrane binding of the nonhydrolyzable analogue of GTP, guanosine-

phate ([35S]GTPgS) [55,83-87]. [35S]GTPgS addition results in accumulation of a stable Ga-[35S]GTPgS complex in spinal cord membranes. Using this procedure, both DPDPE and [D-Ala2]deltorphin II produce a robust stimulation of [35S]GTPgS binding in membranes of the mouse spinal cord [88]. These effects are reversed by delta opioid receptor antagonists. The levels of [35S]GTPgS binding stimulated by DPDPE and [D-Ala2]deltorphin II in membranes of the spinal cord obtained from both heterozygous and homo-zygous MOR-1 knockout mice are similar to those found in wild-type mice [88]. Homozygous DOR-1 knockout mice display markedly reduced spinal antinociception by delta opioid receptor agonists [59]. These data strongly support the idea that the spinal delta opioid receptor is functionally coupled to G protein, and the activation of this G protein-associated receptor by agonists can produce spinal antinociception.

Identification of the G protein a subunits coupled to a specific receptor subtype is a complex process and often requires a number of approaches. Some receptors interact with a multitube of different a subunits, making identification even more difficult. The most straightforward approach is to isolate the receptor/G proteins complex and identify the a subunit component by immunoblotting. However, this is not be easy. Distinct G protein a subunits are thought to be inactivated by pretreatment with toxins, antisera or antisense oligodeoxynucleotides, and the subsequent loss of function assessed. This approach is particularly useful when studying native receptor-mediated functions in vivo and in vitro. Recently, antisense approach is used to explore the G protein a subunits responsible for transducing delta opioid receptor-mediated antinociception [89]. Mice receiving antisense oligodeoxynucleotides to Gia1, Gia2, Gia3, Goa, Gsa, Gqa, or Gx/za subunits show an impaired antinociceptive response to spinal delta opioid receptor agonists [90]. These findings support the idea that spinal delta opioid receptor can interact Gia1, Gia2, Gia3, Goa, Gsa, Gqa, or Gx/za subunits to produce spinal antinociception. As well as the G protein a subunits, we found in the preliminary study that hg subunit is also implicated in the delta opioid receptor-mediated spinal antinociception (unpublished observation).

The interaction of delta opioid receptor-selective ligands with its respective receptor has been reported to hyperpolarize neurons. Hyperpola-rization of neurons in the delta opioid-sensitive pathway may play a major role in the antinociception produced by delta opioids [91-96]. The K + channels represent the largest and most diverse group of any ion channel family that has been identified in cells. Delta opioid receptor agonists have been found to hyperpolarize the membrane potential [91,97-102] and reduce action potential duration in many different neurons [103,104]. The hyper-polarization is accompanied by an increase in resting membrane conductance, varies with changes in extracellular K + concentration and can be blocked by Cs+ and/or Ba + , two cations known to block K+ channels [91,99]. DPDPE reduces the duration of action potentials and decreases voltage-dependent outward K+ currents in both cultured DRG [105] and F11 (a neuroblastoma x DRG hybrid) neurons [106]. These results suggest that delta opioid receptor agonists exert their effects by opening K+ channels.

To determine which K+ channels are opened by opioid agonists, opioid-evoked K+ currents have been studied under the voltage clamp condition. In the submucosal plexus with delta opioid receptors, delta opioids increase a K+ current that has an inwardly rectifying voltage dependence [93,107]. In submocosal neurons, [Met5]enkephalin increases the opening probability of background single K+ channels of small (30-65 pS), intermediate (120-160 pS), and large (220-260 pS) conductance [108].

The spinal antinociceptive effect of DPDPE is blocked totally by a small conductance Ca2 + -activated K+ channel blocker apamin [109]. The DPDPE-induced spinal antinociception is not inhibited by a ATP-sensitive K + channel blocker glyburide [109]. Like morphine, tetraethylammonium, 4-aminopyridine, and charybdotoxin are unable to block the effects of DPDPE [109]. These findings suggest that the modulation of apamin-sensitive K + channels appears to play a role in the DPDPE-induced antinociception in the spinal cord.

The ensuing studies of opioid actions on Ca2+ currents under the voltage clamp condition have proved that opioids exert many of their inhibitory effects by blocking voltage-dependent Ca2+ channels. Activation of delta opioid receptors has been found to reduce mostly N-type Ca2 + currents [110-117]. It should be noted that spinal antinociception induced by the delta opioid receptor agonist is potentiated by a selective N-type Ca2 + channel blocker omega-conotoxin GVIA, whereas the effect of mu opioid receptor agonist is not changed by this treatment [118]. Activation of delta opioid receptors sometimes reduces T-type Ca2+ currents [114,115]. The cloned delta opioid receptor expressed in GH3 cells has been shown to voltage-dependently couple through Gi protein to L-type Ca2+ channels [119]. In submucosal neurons, activation of the delta opioid receptors affects both inwardly rectifying K+ channels and N-type Ca2+ channels in the same neuron [120]. Taken together, it is likely that activation of delta opioid receptors sometimes reduces N-type Ca2+ currents via the opening apamin-

Anti nociception

Figure 1 Role of ion channels in the delta opioid receptor agonist-induced spinal antinociception. Delta opioid receptor agonists acutely inhibit some neurons by increasing the conductance of an apamin-sensitive inwardly rectifying K + channel and decreasing an N-type Ca2 + channel-dependent inward current via activation of Ga and Ghg. These changes, induced by activation of delta opioid receptor, lead to the delta opioid receptor agonist-induced spinal antinociception.

Anti nociception

Figure 1 Role of ion channels in the delta opioid receptor agonist-induced spinal antinociception. Delta opioid receptor agonists acutely inhibit some neurons by increasing the conductance of an apamin-sensitive inwardly rectifying K + channel and decreasing an N-type Ca2 + channel-dependent inward current via activation of Ga and Ghg. These changes, induced by activation of delta opioid receptor, lead to the delta opioid receptor agonist-induced spinal antinociception.

sensitive K+ channel (small conductance Ca2+ activated K+ channel), resulting in the expression of spinal antinociception (Fig. 1).

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