Treatment with antisense oligodeoxynucleotide to the opioid delta receptor selectively inhibits delta 2-agonist antinociception

Using approaches emphasizing differential antagonism of receptor selective agonists and cross-tolerance paradigms, evidence in vivo has suggested the existence of subtypes of opioid delta receptors, which have been termed delta 1 and delta 2. Recent work has elucidated the structure of an opioid delta receptor. The present investigation attempted to continue to test the hypothesis of subtypes of delta receptors and to correlate the cloned delta receptor with the existing pharmacological classification. Synthetic oligodeoxynucleotides (oligos) complementary to the 5' end of the cloned delta receptor coding region (antisense) or its corresponding sequence (sense) were given by intracerebroventricular (i.c.v.) administration to mice, twice-daily for 3 days and antinociceptive responses to selective agonists at putative delta 1 and delta 2 receptors were subsequently determined. Treatment with antisense, but not sense, oligo significantly inhibited the response to [D-Ala2,Glu4]deltorphin (delta 2 agonist), but not to [D-Pen2,D-Pen5]enkephalin (DPDPE, delta 1 agonist). Further, subsequent administration of DPDPE elicited a full antinociceptive response in the same antisense oligo treated mice which did not show a significant response to [D-Ala2,Glu4]deltorphin while antisense oligo treated mice which responded to DPDPE did not show antinociception when tested subsequently with [D-Ala2,Glu4]deltorphin. The data suggest that the cloned delta receptor corresponds to that pharmacologically classified as delta 2 and continue to support the concept of subtypes of opioid delta receptors.     

Molecular models of two competitive inhibitors, IL-2delta2 and IL-2delta3, generated by alternative splicing of human interleukin-2

Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by delta 1- but not delta 2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of delta-opioid receptor agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective delta 1-opioid receptor antagonist, but not with naltriben (NTB), a selective delta 2-opioid receptor antagonist. There were no significant differences in the antinociceptive effect of [D-Ala2]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala2]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal delta 1-opioid receptor-mediated antinociception, but are normally responsive to activation of delta 2-opioid receptors.     

Molecular diversity of the calcium channel alpha2delta subunit

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.