Blythe B. Holmes

Characterization of 14,15-Epoxyeicosatrienoyl-Sulfonamides as 14,15-Epoxyeicosatrienoic Acid Agonists: Use for Studies of Metabolism and Ligand Binding

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-activated potassium (BKCa) channels to cause hyperpolarization and relaxation. We developed a series of 14,15-EET agonists, 14,15-EET-phenyliodosulfonamide (14,15-EET-PISA), 14,15-EET-biotinsulfonamide (14,15-EET-BSA), and 14,15-EET-benzoyldihydrocinnamide-sulfonamide (14,15-EET-BZDC-SA) as tools to characterize 14,15-EET metabolism and binding. Agonist activities of these analogs were characterized in precontraced bovine coronary arterial rings. All three analogs induced concentration-dependent relaxation and were equipotent with 14,15-EET. Relaxations to these analogs were inhibited by the BKCa channel blocker iberiotoxin (100 nM), the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoylmethylsulfonamide (10 μM), and abolished by 20 mM extracellular K+. 14,15-EET-PISA is metabolized to 14,15-dihydroxyeicosatrienoyl-PISA by soluble epoxide hydrolase in bovine coronary arteries and U937 cells but not U937 cell membrane fractions. 14,15-EET-P125ISA binding to human U937 cell membranes was time-dependent, concentration-dependent, and saturable. The specific binding reached equilibrium by 15 min at 4°C and remained unchanged up to 30 min. The estimated Kd and Bmax were 148.3 ± 36.4 nM and 3.3 ± 0.5 pmol/mg protein, respectively. These data suggest that 14,15-EET-PISA, 14,15-EET-BSA, and 14,15-EET-BZDC-SA are full 14,15-EET agonists. 14,15-EET-P125ISA is a new radiolabeled tool to study EET metabolism and binding. Our results also provide preliminary evidence that EETs exert their biological effect through a membrane binding site/receptor.

Reticulocyte 15-Lipoxygenase-I Is Important in Acetylcholine-Induced Endothelium-Dependent Vasorelaxation in Rabbit Aorta

Objective—Aortic 15-lipoxygenase (15-LO) metabolizes arachidonic acid (AA) to 15-hydroperoxyeicosatetraenoic acid, which is then converted to the vasodilators 15-hydroxy-11,12-epoxyeicosatrienoic acid and 11,12,15-trihydroxyeicosatrienoic acid. These metabolites contribute to endothelium-dependent relaxations of rabbit aorta to AA and acetylcholine. We investigated the identity of rabbit aortic 15-LO and studied its importance in the regulation of vascular tone. Methods and Results—RT-PCR using 12-lipoxygenase/15-LO specific primers resulted in a 572-bp product with a sequence identical to 15-LO-I from rabbit aorta. A RT-PCR/restriction digest strategy excluded expression of 12-lipoxygenase. Immunoblotting revealed 15-LO-I expression in rabbit endothelial and smooth muscle cells. Aortic homogenates and cytosolic fractions metabolize AA to 15(S)-hydroxyeicosatetraenoic acid and linoleic acid to 13(S)-hydroxyoctadecadienoic acid. This activity was blocked by LO inhibitors. The kinetic characteristics (Michaelis constant of aortic 15-LO is 2.2±0.3 &mgr;mol/L for AA and 23.5±3.3 &mgr;mol/L for linoleic acid) of aortic 15-LO were similar to those of the purified 15-LO-I. An antisense oligonucleotide inhibited 15-LO-I expression in rabbit aorta. Indomethacin and nitro-l-arginine-resistant relaxations to acetylcholine were inhibited by 15-LO-I antisense oligonucleotide but not by the scrambled oligonucleotide. Conclusions—15-LO-I is expressed in rabbit aortic endothelium and is important in endothelium-dependent regulation of vascular tone.

[D-Pen2-D-Pen5]enkephalin, a delta opioid agonist, given intracerebroventricularly in the mouse produces antinociception through mediation of spinal GABA receptors

Intracerebroventricular (ICV) administration of [D-Pen2-D-Pen5]enkephalin (DPDPE), a delta opioid receptor agonist, activates a descending antinociceptive pathway that inhibits the tail-flick response in mice. Involvement of spinal GABA receptors in this response was studied by giving GABA antagonist intrathecally. First, antinociception produced by intrathecally administered isoguvacine, a GABAA agonist, was inhibited by intrathecal bicuculline (GABA receptor antagonist) or picrotoxin (chloride channel antagonist). Then, antinociception induced by ICV DPDPE was antagonized by intrathecal picrotoxin and bicuculline in a dose-and time-dependent manner. Second, intrathecal administration of 2-hydroxysaclofen, a GABAB antagonist (which inhibited antinociception induced by a GABAB agonist, baclofen, given IT), produced a shift of the dose-response curve for ICV DPDPE to the right. GABAA agonist, baclofen, given IT), produced a shift of the dose-response curve for ICV DPDPE to the right. GABAA and B antagonists given together intrathecally produced a greater than additive antagonistic effect against ICV DPDPE-induced antinociception. Thus, the delta agonist action of DPDPE in the brain leads to activation of descending spinal pathways which involve mediation by spinal GABAA and GABAB receptors in the antinociceptive response.

Inhibiting a Spinal Dynorphin A Component Enhances Intrathecal Morphine Antinociception in Mice

Morphine given intracerebroventricularly releases spinal dynorphin A (Dyn) in mice. The present study was undertaken to determine whether morphine given intrathecally (IT) released Dyn. We demonstrated that the antinociceptive action of morphine was enhanced by procedures that are known to attenuate Dyn action. First, coadministration of the opiate antagonists, naloxone (5 fg), norbinaltorphimine (5 fg) or beta-funaltrexamine (0.25 ng) with IT morphine (0.15 microgram, 5 min) increased antinociceptive percentage maximum possible effect (%MPE) from 30% to 65%. Second, dynorphin antiserum (5 micrograms, 1 h, IT), which neutralizes Dyn action, enhanced morphine (0.2 microgram, 5 min, IT) action; MPE of 27% was increased to 60%. Third, production of desensitization to the antagonistic action of Dyn, IT, by pretreatment with morphine [10 mg/kg, 3 h, subcutaneously (SC)], or 2 micrograms, 3 h, IT) or Dyn (1 ng, 1 h, IT) increased the 30% MPE of IT morphine to 60%. Naloxone [1 ng/kg, intraperitoneally (IP)] enhanced IT morphine at a peak time of 20 min. Nalmefene [1 to 100 ng/kg, per os (PO)] enhanced IT morphine action. In conclusion, the present study showed that IT morphine putatively released spinal Dyn.

Opioid antagonists: indirect antagonism of morphine analgesia by spinal dynorphin A

Naloxone and norbinaltorphimine when given ICV to mice can antagonize IT morphine-induced analgesia indirectly by releasing spinal dynorphin A(1-17) (Dyn A). Dyn A produces an antianalgesic action against IT morphine. In the present study, drugs with varying amounts of opioid antagonist to agonist action (nalbuphine, levallorphan, naltrexone, and naltrindole) were given ICV to determine whether they antagonized IT morphine-induced inhibition of the tail-flick response as an indication of spinal Dyn A release. Additional pharmacological tests were used as criteria for Dyn A release: a) Small doses of the opioid antagonists naloxone and norbinaltorphimine administered IT inhibited the antagonistic action; b) dynorphin antiserum given IT blocked the action of Dyn A; c) desensitization to the effect of Dyn A was produced by 3-h pretreatment with morphine, 10 mg/kg SC, or by pretreatment with the agents themselves. When given ICV, nalbuphine, levallorphan, and naltrexone released Dyn A in the spinal cord to produce an antianalgesic effect. Naltrindole, a delta-receptor antagonist, did not release Dyn A. Dyn A release did not appear to involve delta-receptors. Thus, a number of opioid antagonists inhibit the analgesic action of opioid agonists indirectly through Dyn A release.

Heroin Acts on Delta Opioid Receptors in the Brain of Streptozocin-Induced Diabetic Rats

Abstract Heroin, like morphine, given intracerebroventricularly produces analgesia by acting on μ opioid receptors in most mice. In contrast, in Swiss Webster mice, heroin has the unusual property of acting on brain δ opioid receptors whereas morphine still acts on μ receptors. The literature indicates that in diabetic mice and rats, the μ agonist potency of morphine is diminished while that to a δ receptor agonist is enhanced. The purpose of the present study was to determine if the response to heroin occurred through a δ receptor in the brain of streptozotocin-induced diabetic Sprague-Dawley rats. One week after a cannula was surgically implanted in the lateral ventricle, diabetes was induced by intravenous administration of 55 mg/kg of streptozotocin. Three days later the receptor selectivity of intraventricular heroin in the tail flick test was determined by coadministration of opioid antagonists. In nondiabetic rats, a rightward shift in the dose response curve for heroin was produced by naloxone. D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2, a more μ receptor selective antagonist given in a single dose experiment, also inhibited heroin action. Thus, heroin acted on μ receptors. In diabetic rats, intracerebroventricular naltrindole, but not naloxone nor CTOP, inhibited the heroin response and indicated a δ agonist action for heroin. Inhibition by intrathecal yohimbine of the μ (nondiabetic) and bicuculline of the δ response (diabetic) suggested spinal α2-adrenergic and GABAA receptor mediation, respectively, for the descending systems. In conclusion, the response to heroin was changed from μ in nondiabetic rats to a δ receptor action in diabetic rats. Understanding the basis for this change in receptor selectivity of heroin could provide an important avenue for investigating determinants of opioid receptor function.

Determination of δ-opioid receptors in NG108-15 cells

The delta-opioid receptors in mouse neuroblastoma x rat glioma NG108-15 cells were characterized by receptor binding and cAMP assays. Saturation binding assays using [3H][D-Pen5]enkephalin (DPDPE) or [3H][D-Ser2, Leu5, Thr6]enkephalin (DSLET) gave similar binding capacities (Bmax). Competition binding assays showed that DPDPE and DSLET have similar affinity for the [3H]DPDPE or 3[H]DSLET binding sites. The rank order of potency of competition with [3H]DPDPE and [3H]DSLET was similar: naltriben approximately DSLET > or = DPDPE > 7-benzylidenenaltrexone (BNTX). Both DPDPE and DSLET were found to decrease cAMP formation. The action of DSLET was antagonized by naltriben but not BNTX, while the action of DPDPE was reversed by both antagonists. Therefore, the delta-opioid receptor in NG108-15 cells has similar affinity for the agonists DPDPE and DSLET, and a higher affinity for the antagonist naltriben than BNTX.

Supraspinal flumazenil inhibits the antianalgesic action of spinal dynorphin A (1-17)

DynorphinA (Dyn) administered intrathecally or released spinally in mice produces antianalgesia, that is, antagonizes morphine analgesia (tail-flick test). Spinal transection eliminates this Dyn antianalgesia. Present results in mice show that intracerebroventricular administration of flumazenil, a benzodiazepine receptor antagonist, also eliminated the antianalgesic action of Dyn; flumazenil in the brain eliminated the suppressant effect of intrathecal Dyn on intrathecal and intracerebroventricular morphine-induced antinociception. Intracerebroventricular clonidine, naloxone, and norbinaltorphimine release spinal Dyn. The latent antinociceptive actions of these compounds were uncovered by intracerebroventricular flumazenil. Thus, Dyn, given intrathecally or released spinally, activates a pathway that is inhibited by intracerebroventricular flumazenil. Dyn antianalgesia is not significantly altered by intracerebroventricular administration of bicuculline and picrotoxin, suggesting that activation of the gamma-aminobutyric acid receptor has little if any involvement in the antianalgesic action of Dyn. The antagonistic effect of Dyn seems to be mimicked by benzodiazepine agonists. Furthermore, administration of a benzodiazepine receptor inverse agonist (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) inhibited Dyn antianalgesia as did flumazenil. Thus, flumazenil, through a benzodiazepine antagonist or inverse agonist action, interrupts, as does spinal transection, the neuronal circuit (cord/brain/cord) necessary for the antianalgesic action of spinal Dyn. Because Dyn antianalgesia is an indirect action, activation of the neuronal circuit must lead to the release of a direct-acting antianalgesic mediator in the spinal cord.