Rebecca P. Martinez

G-protein antisense oligodeoxyribonucleotides and μ-opioid supraspinal antinociception

The present study utilized an in vivo antisense strategy to examine the functional interaction of supraspinal mu-opioid receptors with the G protein subunits Gi1 alpha, Gi2 alpha, Gi3 alpha and Gs alpha. Mice were injected intracerebroventricularly (i.c.v.) with 33-base phosphorothioate oligodeoxyribonucleotides (12.5 micrograms) or with vehicle in equal volume (sterile water, 5 microliters) and the antinociceptive responses to i.c.v. morphine [D-Ala2,NMePhe4,Gly5-ol]enkephalin (DAMGO) or sufentanil were determined 18-24 h later using the tail-flick test. Treatment with antisense to Gi2 alpha, but not the other subunits, significantly attenuated morphine-induced antinociception. The degree of attenuation for the three mu-opioid agonists was in the order morphine > DAMGO > sufentanil, the inverse of their intrinsic efficacy.

The ‘glibenclamide-shift’ of centrally-acting antinociceptive agents in mice

Morphine-induced antinociception is antagonized by the K(+)-channel blocker glibenclamide (glyburide; Glib), implicating ATP-sensitive (KATP) K+ channels in the analgesic effect of opioids. The present study examined the generality of this conclusion by measuring the effect of Glib on supraspinal (intracerebroventricular; i.c.v.) antinociception produced by representative mu-opioids and the non-opioids pilocarpine and two alpha 2-adrenoceptor agonists (clonidine and tizanidine) using the mouse tail-flick test. Concurrent administration of Glib (40 micrograms, i.c.v.) produced a significant rightward shift of the dose-response curve of morphine, levorphanol, methadone, pilocarpine, clonidine and tizanidine; a modest, but not statistically significant, rightward shift of the dose-response curves of the mu-selective peptides DAMGO ([D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin) and PL017 ([N-Me-Phe3,D-Pro4]-morphiceptin); and no shift of the dose-response curves of alfentanil, carfentanil, fentanyl, sufentanil, or beta-endorphin. Glib produced a leftward shift of the dose-response curve of etorphine. These data support the involvement of KATP-type K+ channels in mediation of supraspinal antinociception, differentiate Glib-sensitive and Glib-insensitive opioid agonists, and reveal fundamental differences among antinociceptive agents in the extent of demonstrable utilization of this transduction pathway.

Endothelin-1-induced nociception

Intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of morphine to mice antagonized the abdominal constriction induced by an i.p. injection of endothelin-1 (ET-1; 0.1 mg/kg). The ED50 values (95% confidence intervals) were 39.3 (16.5-80.2) ng and 1.5 (0.8-4.9) ng, respectively. The antagonism of ET-1-induced abdominal constriction by morphine was blocked by naloxone (1.0 mg/kg, s.c.) or by 24 h pretreatment with beta-funaltrexamine (beta-FNA; 8.84 micrograms, i.c.v.). These results demonstrate for the first time that the stimulus resulting from an i.p. injection of ET-1 is transmitted via ascending (pain) pathways that are subject to attenuation by opioid (mu) receptor activation. Hence, ET-1-induced abdominal constriction is a new pain model which, given the other pharmacology of ET-1, might represent a unique model with potential specific utility for anginal or other visceral pain.

Morphine antinociception is mediated through a LiCl-sensitive, IP3-restorable pathway

Pretreatment (18 h) of mice with a single s.c. injection of LiCl (10 mmol/kg) reduced the antinociceptive action of centrally administered (i.c.v.) morphine in the tail-flick test (ED50 = 7.7 micrograms) compared to vehicle-treated controls (ED50 = 1.8 micrograms). The coadministration of inositol-1,4,5-trisphosphate (IP3; 20 micrograms) with morphine restored the morphine-induced antinociception (ED50 = 2.4 micrograms) in LiCl-pretreated mice to control levels. These finding implicate a LiCl-sensitive (possibly phosphoinositide) second messenger pathway in the mediation of morphine-induced analgesia.

Lack of antinociceptive efficacy of intracerebroventricular [D-Ala2,Glu4]deltorphin, but not [D-Pen2,D-Pen5]enkephalin, in the μ-opioid receptor deficient CXBX mouse

The antinociceptive efficacy of [D-Pen2,D-Pen5]enkephalin (DPDPE) (delta 1 agonist) and [D-Ala2,Glu4]deltorphin (delta 2 agonist) was evaluated following intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration in CD-1 and CXBK strains of mice using the radiant heat tail-flick test. Following i.c.v. administration, [D-Ala2,Glu4]deltorphin was effective in CD-1, but not CXBK, mice; DPDPE was approximately equiactive in both strains. While i.c.v. [D-Ala2,Glu4]deltorphin did not produce antinociception in the CXBK mouse, it effectively antagonized the antinociceptive actions of i.c.v. DPDPE. [D-Ala2,Glu4]deltorphin was effective following i.t. administration in both strains. These data suggest possible differences in the supraspinal populations of opioid delta receptor subtypes in the CXBK strain. On the basis of previously established selectivity of these agonists, the CXBK mouse may have a predominate population of supraspinal opioid delta 1, rather than delta 2, receptors.

Opioid efficacy is linked to the LiCl-sensitive, inositol-1,4,5-trisphosphate-restorable pathway

Previous work demonstrated that a single subcutaneous injection of LiCl (10 mmol/kg; 18 h prior) significantly reduces the antinociceptive action of centrally administered (intracerebroventricular; i.c.v.) morphine in the tail-flick and that inositol-1,4,5-trisphosphate (IP3; 20 micrograms) restores the morphine response in LiCl-pretreated mice, implicating a phosphoinositide second messenger pathway in the mediation of morphine-induced analgesia. We now report that LiCl pretreatment shifted the antinociceptive dose-response curve produced by the opioid agonists morphine, [D-Ala2, MePhe4, Gly5-ol]enkephalin (DAMGO) and sufentanil in inverse order of their intrinsic efficacy. These results implicate the phosphoinositide pathway(s) as important determinant(s) of opioid efficacy and are interpreted as evidence for the existence of a second-messenger reserve for opioids of high efficacy and the possible role of the phosphoinositide pathway in the development of morphine tolerance.

A Gi2α antisense oligonucleotide differentiates morphine antinociception, constipation and acute dependence in mice

In the same mice in which the intracerebroventricular (i.c.v.) administration of antisense oligodeoxyribonucleotide (oligo) directed against the Gi2alpha (but not Gi1alpha, Gi3alpha or G(s)alpha) G-protein subunits attenuated i.c.v. morphine-induced antinociception in the tail-flick test, none of the oligos altered naloxone-precipitated jumping (acute dependence). Likewise, none of the oligos significantly altered morphine-induced constipation. Hence, i.c.v. morphine-induced antinociception might be preferentially mediated via transduction pathway(s) different from constipation or acute dependence, offering novel opportunities for drug discovery.

The Novel, Orally Active, Delta Opioid RWJ-394674 Is Biotransformed to the Potent Mu Opioid RWJ-413216

Although the mu opioid receptor is the primary target of marketed opioid analgesics, several studies suggest the advantageous effect of combinations of mu and delta opioids. The novel compound RWJ-394674 [N,N-diethyl-4-[(8-phenethyl-8-azabicyclo]3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide]; bound with high affinity to the delta opioid receptor (0.2 nM) and with weaker affinity to the mu opioid receptor (72 nM). 5′-O-(3-[35S]-thio)triphosphate binding assay demonstrated its delta agonist function. Surprisingly given this pharmacologic profile, RWJ-394674 exhibited potent oral antinociception (ED50 = 10.5 μmol/kg or 5 mg/kg) in the mouse hot-plate (48°C) test and produced a moderate Straub tail. Antagonist studies in the more stringent 55°C hot-plate test demonstrated the antinociception produced by RWJ-394674 to be sensitive to the nonselective opioid antagonist naloxone as well as to the delta- and mu-selective antagonists, naltrindole and β-funaltrexamine, respectively. In vitro studies demonstrated that RWJ-394674 was metabolized by hepatic microsomes to its N-desethyl analog, RWJ-413216 [N-ethyl-4-[(8-phenethyl-8-azabicyclo[3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide], which, in contrast to RWJ-394674, had a high affinity for the mu rather than the delta opioid receptor and was an agonist at both. Pharmacokinetic studies in the rat revealed that oral administration of RWJ-394674 rapidly gave rise to detectable plasma levels of RWJ-413216, which reached levels equivalent to those of RWJ-394674 by 1 h. RWJ-413216 itself demonstrated a potent oral antinociceptive effect. Thus, RWJ-394674 is a delta opioid receptor agonist that appears to augment its antinociceptive effect through biotransformation to a novel mu opioid receptor-selective agonist.

Etonitazene-induced antinociception in μ1 opioid receptor deficient CXBK mice : evidence for a role for μ2 receptors in supraspinal antinociception

The prevailing view is that supraspinal mu opioid-mediated antinociception in mice is mediated via the mu 1 subtype. The purpose of the present study was to determine if the highly mu-selective compound etonitazene could produce supraspinal (intracerebroventricular; i.c.v.) antinociception in CXBK mice, which are deficient in brain mu1, but not mu2, opioid receptors. CXBK or normal Crl:CD-1 (ICR)BR mice were administered graded doses of etonitazene i.c.v. and 15 min later antinociception was assessed by a standard radiant-heat or 55 degrees C water tail-flick test. Etonitazene produced dose-related antinociception that was blocked by naloxone and by beta-FNA (demonstrating a mu opioid mechanism), but not by either ICI-174,864 or naltrindole (demonstrating the lack of involvement of delta opioid receptors). These findings suggest that mu2 opioid receptors are important contributors to opioid-induced supraspinal antinociception in mice.

Antinociceptive defect of beige-J mice reversed by i.c.v. IP3 or myo-inositol.

The C57BL/6J-bgJ/bgJ (beige-J) mutation imparts a blunted response to intracerebroventricular (i.c.v.) morphine in the tail-flick test, without altered micro-opioid receptor number or morphine affinity. We now report that co-administration of IP3 (36.1 nmol) restored morphine responsiveness of beige-J mice to essentially that of normal littermates (bg+/bg-; ED50 = 3.9 and 3.5 nmol, respectively). IP3 had no effect on morphine-induced antinociception in control animals. Neither myoinositol at 36.1 nmol nor IP6 at the highest testable dose (4.5 nmol) had a similar effect. Myo-inositol at 5.5 mumol restored beige-J responsiveness to that of littermates. These findings implicate some component of the phosphoinositide cycle in the antinociceptive defect of beige-J mice.