Regulation of hsp expression during rodent spermatogenesis

As part of an effort to develop peptides with selective kappa-opioid antagonist activity, a series of N-alkylated [D-Pro10]dynorphin A-(1-11) derivatives were made through solid-phase peptide synthesis: R-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-D-Pro-LysOH, where R = N-benzyl, N-cyclopropylmethyl, N,N-dicyclopropylmethyl, or N,N-diallyl. These derivatives and dynorphin A-(1-13)NH2 were evaluated for kappa-opioid receptor binding affinity and potency as inhibitors of adenylyl cyclase. Equilibrium competition binding experiments using [3H]diprenorphine (approximately 600 pM) were performed on membranes prepared from cultured Chinese hamster ovary (CHO) cells stably expressing the rat kappa-opioid receptor. Tissue prepared from this cell line was used to evaluate opioid peptide inhibition of forskolin-stimulated (50 microM) adenylyl cyclase activity. Displacement of [3H]diprenorphine specific binding by these peptides was observed with a rank order of affinity (Ki, nM) = [D-Pro10]dynorphin A-(1-11) (0.13) > dynorphin A-(1-13)NH2 (0.34) > N-cyclopropylmethyl- (1.4) > N,N-dicyclopropylmethyl- (12.6) approximately N-benzyl- (18.3) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11) (26.0). A similar rank order was observed for potency of adenylyl cyclase inhibition (IC50, nM): [D-Pro10]dynorphin A-(1-11) (0.12) approximately dynorphin A-(1-13)NH2 (0.19) > N-cyclopropylmethyl- (2.7) > N,N-dicyclopropylmethyl- (13.2) approximately N,N-diallyl- (18.0) approximately N-benzyl-[D-Pro10]dynorphin A-(1-11) (36.4). The peptides differed in their percent maximal inhibition of adenylyl cyclase activity: dynorphin A-(1-13)NH2 (100%) approximately N-cyclopropylmethyl- (94.3%) approximately [D-Pro10]dynorphin A-(1-11) (87.9%) > N-benzyl- (71.4%) > N,N-dicyclopropylmethyl- (23.6%) approximately N,N-diallyl-[D-Pro10]dynorphin A-(1-11)(18.9%). As the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) derivatives were found to have only weak partial agonist activity with respect to adenylyl cyclase inhibition, they were evaluated for their ability to reverse dynorphin A-(1-13)NH2 (10 nM) inhibition of adenylyl cyclase activity. N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) reversed dynorphin A-(1-13)NH2 inhibition to levels equal to the maximal inhibition produced by N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) alone. This weak partial agonism combined with nanomolar potency render the N,N-dicyclopropylmethyl- and N,N-diallyl-[D-Pro10]dynorphin A-(1-11) compounds promising leads for further attempts to synthesize peptide kappa-opioid receptor antagonists.     

Kappa1- and kappa2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional kappa-opioid receptor subtypes in rat brain. 2. The kappa receptor agonists (-)-ethylketocyclazocine ((-)-EKC), U69593 and the endogenous opioid peptide dynorphin A1-13 caused a naloxone-reversible inhibition of NMDA-induced [3H]-dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the mu agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and the delta agonist D-Pen2-D-Pen5-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1-13, U69593 and (-)-EKC on NMDA-induced [3H]-dopamine release were not changed by the delta1/delta2-opioid receptor antagonist naltrindole (up to a concentration of 1 microM, but reversed by the kappa receptor antagonist nor-binaltorphimine (nor-BNI), with an IC50) as low as 0.02 nM, indicating the involvement of U69593-sensitive kappa1-opioid receptors. 3. NMDA-induced [14C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD2 about 7.2), dynorphin A1-13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1-13 and (-)-EKC, on NMDA-induced [14C]-ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of delta-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1-13 and (-)-EKC on [14C]-ACh release were readily antagonized by nor-BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593-insensitive kappa2-opioid receptors in the inhibitory effects of dynorphin A1-13 and (-)-EKC. 4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative kappa3-opioid receptor, antagonized the inhibitory effects of dynorphin A1-13 and (-)-EKC on [3H]-dopamine and [14C]-ACh release as well as that of U69593 on [3H]-dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case. 5. In conclusion, whereas activation of kappa1-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, kappa2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct kappa-opioid receptor subtypes in the brain.     

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) > Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.     

Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships

The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol(-1). Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nM and Bmax of 94 fmol mg(-1) of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs = 0.104 min(-1); K1 =0.034 min(-1): T1/2=20 min; K(+1)=0.07 min nM(-1). Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(delta), OP2(kappa) and OP3(mu). Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)-NH2([F/G]NC(1-13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH = NC-NH2-NC(1-13)-NH2 > > NC(1-12)-NH2 > NC(1-13)-OH > > NC(1-11)-NH2, and for NC and NC(1-13)-NH2 analogues: [Tyr1]NC-NH2 > or = [Leu1]NC(1-13)-NH2 > or = [Tyr1]NC(1-13)-NH2 > or = [F/G]NC(1-13)-NH2 > > [Phe3]NC(1-13)-NH2 > [DF/G]NC(1-13)-NH2. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 microM indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor.     

Mechanism of the dynorphin-induced dualistic effect on free intracellular Ca2+ concentration in cultured rat spinal neurons

In order to study the different mechanisms of dynorphin spinal analgesia and neurotoxicity at low and high doses, the effects of various concentrations of dynorphin A-(1-17) on the free intracellular Ca2+ concentration ([Ca2+]i) in the cultured rat spinal neurons were studied using single cell microspectrofluorimetry. While dynorphin A-(1-17) 0.1-100 microM had no significant effect on basal [Ca2+]i, dynorphin A-(1-17) 0.1 and 1 microM significantly decreased the high KCl-evoked peak [Ca2+]i by 94% and 83% respectively. Dynorphin A-(1-17) 10 and 100 microM did not affect the peak [Ca2+]i following K+ depolarization, but in all these neurons there was a sustained and irreversible rise in [Ca2+]i following high-K+ challenge. Pretreatment with the specific kappa-opioid receptor antagonist nor-binaltorphimine 10 microM, but not the competitive NMDA receptor antagonist, DL-2-amino-5-phosphonovalerate (APV) 10 microM, significantly blocked the inhibitory effect of dynorphin A-(1-17) 0.1 microM on peak [Ca2+]i. However, APV 10 microM and nor-binaltorphimine 10 microM significantly antagonized the sustained rise in [Ca2+]i induced by a high concentration of dynorphin A-(1-17) 10 microM. Furthermore, in the presence, and following the addition, of increasing concentrations of dynorphin A-(1-17) (0.1, 1, 10 and 100 microM), the high concentrations of dynorphin A-(1-17) failed to produce a sustained rise in peak [Ca2+]i. These results suggested that dynorphin exerted a dualistic modulatory effect on [Ca2+]i in cultured rat spinal neurons, inducing a sustained and irreversible intracellular Ca2+ overload via activation of both NMDA and kappa-opioid receptors at higher concentrations, but inhibiting depolarization-evoked Ca2+ influx via kappa-opioid but not NMDA receptors at lower concentrations. Serial addition of graded concentrations of dynorphin A-(1-17) prevented the effect of high concentrations of dynorphin A-(1-17) on [Ca2+]i.     

Parathyroid hormone (PTH) stimulates adrenocorticotropin release in AtT-20 cells stably expressing a common receptor for PTH and PTH-related peptide

Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.     

Ligand binding profiles of U-69, 593-sensitive and-insensitive sites in human cerebral cortex membranes: evidence of kappa opioid receptors heterogeneity

Receptor binding studies were performed to characterize the properties of subtypes of kappa opioid receptors in membrane preparations of human cerebral cortex. [3H]U69,593 ([3H]U69), a selective kappa 1-agonist, and [3H]diprenorphine ([3H]DIP), a non-selective opioid antagonist, in the presence of 1 microM each of DAMGO, DPDPE and U-69 to block mu-, delta-, and kappa 1-sites, labeled single population of binding sites, respectively. [3H]U-69 binding sites (KD = 3.8 +/- 0.2 nM, Bmax = 6.3 +/- 0.2 fmol/mg protein) had a binding profile that correspond to kappa 1-receptor. That is, dynorphin A (1-13) (Dyn A), bremazocine (BZC), U50,488H (U50), (-)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [3H]DIP labeled binding sites (Kd = 7.3 +/- 0.2 nM, Bmax = 102 +/- 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that kappa 1 and Kappa 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles.     

Logistic regression analysis of twin data: estimation of parameters of the multifactorial liability-threshold model

This study with the rat evaluated the contribution of omega-conotoxin GVIA-(omega-CgTx) and verapamil-sensitive Ca2+ channels in behavioural, antinociceptive and thermoregulatory responses to intracerebroventricular (i.c.v.) injection of [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) and dynorphin A-(1-17), which are selective agonists for putative mu, delta and kappa-opioid receptors, respectively. The rats treated with omega-CgTx (8-32 pmol i.c.v.) showed transient, dose-dependent shaking behaviour, hyperalgesia and hypothermia which gradually disappeared within 4 h. The behaviour of the rats was normal by 24 h. Histological examination of brain sections showed morphological alterations of neurons in the hippocampus, medial-basal hypothalamus and pyriform cortex. antinociception, catalepsy and thermoregulatory responses elicited by DAMGO (0.4 and 2.0 nmol) were significantly prolonged and potentiated by verapamil (20 pmol i.c.v. 15 min before) or omega-CgTx (8 pmol 24 h before). Antinociception and hypothermia induced by DPDPE were antagonized by verapamil and omega-CgTx, whereas only omega-CgTx prevented the behavioural arousal observed after DPDPE. Similarly, hypothermia induced by dynorphin A-(1-17) (5.0 nmol) and by the kappa-opioid receptor agonist U50,488H (215 nmol) was antagonized by the two Ca2+ channel blockers but only omega-CgTx prevented the barrel rolling and bizarre postures caused by the opioid peptide.     

Dynorphin A(6-12) analogs suppress thermal edema

Dynorphin A (Dyn A) is a 17-residue opioid peptide derived from prodynorphin precursors found in mammalian tissues. Removal of Tyr1 from Dyn A produces a peptide that is more potent than Dyn A in attenuating the acute phase of the inflammatory response, as measured by inhibition of heat-induced edema in the anesthetized rat's paw (exposure to 58 degrees C water for 1 min). Dyn A(2-17), however, no longer interacts with opioid receptors. It was postulated that the non-opioid anti-inflammatory actions of Dyn A(2-17) may reside in Dyn A(6-12); that is, Arg-Arg-Ile-Arg-Pro-Lys-Leu. here we report on the activities of Dyn A(6-12) analogs modified by substitutions on the N terminus, by single N-methyl substitution and by single replacement of residues by alanine. The results indicated that the minimal sequence required for an anti-edema ED50 of <1.0 micromol/kg i.v. was anisoyl-Arg6-Arg7-Xaa8-Arg9-Pro10)-Xaa11-+ ++Xaa12-NH2. A prototype, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, with an ED50 of 0.20 micromol/kg i.v. compared to an ED50 of 0.08 micromol/kg i.v. for Dyn A(2-17), was selected for further tests of biological activity. This analog, like Dyn A(2-17), lowered blood pressure in anesthetized rats. In a model of neurogenic inflammation, produced by antidromic stimulation of the vagus in the anesthetized rat, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, 0.23 micromol/kg i.v., attenuated the negativity of tracheal tissue interstitial pressure (Pif), which normally develops after nerve stimulation. Modulation of interstitial pressure may be the mechanistic basis for the anti-edema properties of these Dyn A(6-12) analogs.     

Classical conditioning in the fetal rat: Reinforcing properties of dynorphin A (1–13).

This study examined the reinforcing properties of dynorphin A (1-13) in a single-trial classical conditioning paradigm in the E20 rat fetus. Injection of dynorphin into the cisterna magna increased fetal motor activity and reduced facial wiping in a test of perioral cutaneous responsiveness. Dynorphin was effective as an unconditioned stimulus (US) in a classical conditioning paradigm using an artificial nipple conditioned stimulus (CS) and dynorphin A (1-13) US. The association between CS and US was dependent on activity in the kappa opioid system. Re-exposure to the artificial nipple CS after a single pairing of the nipple with dynorphin resulted in conditioned activation of the kappa opioid system. Dynorphin A (1-13) functions as a reinforcer for classical conditioning in the rat fetus after intracisternal or intrahemispheric injection with the conditioned response depending on route of administration and site of injection. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Procorticotrophin-releasing hormone: endoproteolytic processing and differential release of its derived peptides within AtT20 cells

Procorticotrophin-releasing hormone (proCRH) is expressed mainly in the hypothalamus and in the placenta, where it undergoes tissue-specific endoproteolysis. Our results show that within stably transfected AtT20/D16V cells proCRH is cleaved to generate two fragments of approximately 8 and 3 kDa which could account for proCRH(125-194) and proCRH(125-151), respectively, and a 4.5 kDa product which could account for mature IR-CRH(1-41). The immunofluorescence staining patterns for IR-CRH and IR-ACTH and their response of secretagogues indicate targeting of proCRH and POMC to the secretory pathway in transfected AtT20 cells. In this work, we have used a unique set of specific RIAs and IRMAs to the full length POMC and proCRH molecules and several products of endoproteolytic processing to assess if they could be released differentially in response to stimulation. Although the release of both IR-ACTH and IR-CRH peptides from transfected AtT20 cells is stimulated in response to exposure to high potassium stimulation (51 mM KCl/SmM CaCl2), the sorting index (SI) suggests that mature ACTH is sorted to the regulated secretory pathway 2.1-fold more efficiently than mature CRH(1-41). Mature ACTH is also sorted to the regulated secretory pathway 9-fold more efficiently than IR-proCRH(125-151). Also, mature CRH(1-41) is sorted to the regulated secretory pathway 3-fold more efficiently than IR-proCRH(125-151). These results therefore indicate that the intracellular mechanisms for the storage and release of POMC, proCRH and their endoproteolytic products differ and would sustain the hypothesis that within mammalian peptidergic cells, different biologically active peptides originating from the same or different precursor molecules, could be differentially released in response to specific stimuli. This would give these cells the capacity to finely regulate neurotransmitter release in response to environmental and physiological demands.     

A study on the effect of dynorphin A- (1-13) ICV injection on water, sodium and potassium excretion of rats

The effect of Kappa-agonist dynorphin A-(1-13) on the urine flow was studied in ethanol-anesthetized rats. Intracerebroventricular injection of dynorphin A- (1-13) (5 micrograms, 10 micrograms) induced a potent diuretic response with insignificant action on mean arterial pressure and heart rate. The diuretic response began to show at about 20 min after injection and lasted 120 min. This diuretic effect could be almost completely blocked by pretreatment with naloxone (20 micrograms) or atropine (10 micrograms).     

Effects of the kappa-opioid dynorphin A(1-13) on learning and memory in mice

The effects of intracerebroventricular administration of dynorphin A(1-13) on scopolamine- and pirenzepine-induced amnesia were investigated in mice by observing the step-down-type passive avoidance response and spontaneous alternation performance. The pre- or post-training, or preretention administration of dynorphin A(1-13) (0.3-10 micrograms) alone failed to affect the passive avoidance response, while scopolamine (1 mg/kg) significantly inhibited it. Dynorphin A(1-13) (1 microgram) given 15 min before training and retention tests, but not immediately after training, significantly improved the scopolamine (1 mg/kg)-induced impairment of passive avoidance response, indicating the anti-amnesic effects of dynorphin A(1-13). A lower dose (1 mg/kg) of the kappa-opioid receptor antagonist (-)-(1R,5R,9R)-5,9-diethyl-2-(3-furyl-methyl)-2'-hydroxy-6,7-benzomorpha n reversed the anti-amnesic effects of dynorphin A(1-13) (1 microgram). In contrast, although dynorphin A(1-13) (1, 3 and 10 micrograms) did not influence spontaneous alternation performance, scopolamine (1 mg/kg) and the muscarinic M1 receptor antagonist pirenzepine (3 micrograms) markedly decreased spontaneous alternation performance. Dynorphin A(1-13) (3, 5.6 and/or 10 micrograms) significantly improved the scopolamine (1 mg/kg)- and pirenzepine (3 micrograms)-induced impairment of spontaneous alternation performance. The improving effects of dynorphin A(1-13) (3 micrograms) were almost completely reversed by pretreatment with nor-binaltorphimine (4 micrograms), a kappa-selective opioid receptor antagonist. These results suggest that the stimulation of kappa-opioid receptors improves memory dysfunctions resulting from the blockade of muscarinic M1 receptors.     

Different domains of the ORL1 and kappa-opioid receptors are involved in recognition of nociceptin and dynorphin A

In order to gain further insight into the functional architecture of structurally related G protein-coupled receptors, the ORL1 (nociceptin) and opioid receptors, we have constructed chimeras of ORL1 and mu-, delta- and kappa-opioid receptors, and compared their binding and functional properties with those of the parent receptors. We find in particular that a ORL1-kappa-opioid (O-K) hybrid construct has retained high affinity for non-type-selective opiate ligands, and has acquired the ability to bind and respond to enkephalins and mu- and/or delta-opioid receptor-selective enkephalins analogs, thus behaving like a 'universal' opioid receptor. Most significantly however, whilst the ORL1 and kappa-opioid receptors display high binding preference (KD 0.1 vs. 100 nM) for their respective endogenous ligands, nociceptin and dynorphin A, the O-K chimeric receptor binds both nociceptin and dynorphin A, with high affinity (KD < 1 nM). Together, these data (i) add weight to the hypothesis that the extracellular loops of opioid receptors act as a filter for ligand selection, and (ii) demonstrate that different domains of the ORL1 and kappa-opioid receptors are involved in recognition of their endogenous peptide ligands.     

Characterization of VIP receptor-effector system antagonists in rat and mouse peritoneal macrophages

In the present study we show that the synthetic peptides [4-Cl-D-Phe6,Leu17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 inhibit in a competitive manner the specific [125I]VIP binding to both rat and mouse peritoneal macrophages. In rat peritoneal macrophages, the order of potency of the different peptides, as expressed by the IC50 values was: VIP (IC50 = 1.90 +/- 0.16 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 125.8 +/- 13.2 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 354.8 +/- 21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC50 = 1.58 +/- 0.12 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 110.8 +/- 10.7 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 251 +/- 19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl-D-Phe6,Leu17]VIP and [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 in rat and mouse peritoneal macrophages was confirmed by: (a) the shift to the right of VIP dose-stimulated cyclic AMP production curves in the presence of the two antagonists; (b) the agreement between the order of efficacy of the two peptides in competition experiments with the corresponding inhibition of cyclic AMP production; (c) the inefficiency of the two antagonists on the stimulation of cyclic AMP production by the beta-adrenoceptor agonist isoproterenol, which indicates the specificity of the interaction; (d) the synergic effect of VIP on isoproterenol-stimulated cyclic AMP production was completely abolished by [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a beta-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2; (e) in cross-linking experiments, the intensity of the labeling of the [125I]VIP/receptor complexes was significantly lower with the antagonists than in the control experimental situation in both mouse and rat peritoneal macrophage membranes.     

Dynorphin A-(1-13) attenuates basal forebrain-lesion-induced amnesia in rats

The effects of dynorphin A-(1-13), an endogenous kappa opioid agonist, on basal forebrain (BF)-lesion-induced amnesia in rats were investigated using step-through-type passive avoidance task. The BF was lesioned by injecting the cholinergic neurotoxin ibotenic acid (6 micrograms/side). The number of rats achieving the cut-off time (600 s) of step-through latency (STL) in BF-lesioned group significantly decreased as compared with that in sham-operated group. Dynorphin A-(1-13) (0.3 micrograms) significantly increased the number of rats achieving the cut-off time of STL in BF-lesioned rats. These results suggest that dynorphins play an improving role in the impairment of memory processes in BF-lesioned rats.     

Molecular modelling of the ORL1 receptor and its complex with nociceptin

The opioid receptor like (ORL1) receptor is a G-protein coupled receptor superfamily, and regulates a plethora of neurophysiological functions. The structural requirements for receptor activation by its endogenous agonist, nociceptin (FGGFTGARKSARKLANQ), differ markedly from those of the kappa-opioid receptor and its putative peptide agonist, dynorphin A (YGGFLRRIRPKLKWDNQ). In order to probe the functional architecture of the ORL1 receptor, a molecular model of the receptor has been built, including the TM domain and the extra- and intracellular loops. An extended binding site able to accommodate nociceptin-(1-13), the shortest fully active analogue of nociceptin, has been characterized. The N-terminal FGGF tetrapeptide is proposed to bind in a highly conserved region, comprising two distinct hydrophobic pockets in a cavity formed by TM helices 3, 5, 6 and 7, capped by the acidic second extracellular (EL2) loop controlling access to the TM elements of the peptide binding site. The nociceptin conformation provides for the selective preference of the ORL1 receptor for nociceptin over dynorphin A, conferred by residue positions 5 and 6 (TG versus LR), and the favourable interaction of its highly positively charged core (residues 8-13) with the EL2 loop, thought to mediate receptor activation. The functional roles of the EL2 loop and the conserved N-terminal tetrapeptide opioid 'message' binding site are discussed in the context of the different structural requirements of the ORL1 and kappa-opioid receptors for activation.     

Tramadol, M1 metabolite and enantiomer affinities for cloned human opioid receptors expressed in transfected HN9.10 neuroblastoma cells

Tramadol hydrochloride is a centrally acting synthetic analgesic in widespread clinical use. Despite different degrees of opioid-like characteristics in preclinical tests, it is characterized by lack of full naloxone reversibility or naloxone-precipitated withdrawal in humans. To investigate this apparent discrepancy, the present study measured the affinity of tramadol (and its enantiomers) and an active O-desmethyl metabolite (M1) (and its enantiomers) to cloned human opioid receptors of the mu, delta and kappa type stably expressed in HN9.10 neuroblastoma cells. At mu sites, the Ki values for tramadol, its (+) and (-) enantiomers, M1, and its (+) and (-) enantiomers were 17000, 15700, 28800, 3190, 153 and 9680 nM, respectively, compared to 7.1 nM for morphine. These results are consistent with the suggestion of a non-opioid contribution to the clinical profile of tramadol.     

Thromboxane A2 stimulated signal transduction in vascular smooth muscle

Thromboxane A2 (TXA2) is a potent, labile vasoconstrictor which stimulates vessel contraction through vascular smooth muscle TXA2 receptors differing from those in platelets. We studied TXA2-stimulated events in cultured adult rat aortic smooth muscle cells. The stable TXA2 mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) competed for TXA2 agonist binding to vascular smooth muscle cells with an IC50 of 10 +/- 1 nM. In fura-2-loaded cells, U46619 increased free cytosolic Ca++ concentration with an EC50 of 49 +/- 14 nM. The increase in free cytosolic Ca++ was rapid, transient and independent of extracellular Ca++ or Ca++ antagonists and thus was due to release from intracellular stores. U46619-mediated Ca++ release was temporally associated with phosphorylation of myosin light chains, increased accumulation of 1,4,5-inositol trisphosphate (EC50 = 32 +/- 4 nM) and cytoplasmic acidification from pH 7.06 +/- 0.01 to 7.00 +/- 0.02 (P = .02). Ca++ release was 53% attenuated by the phospholipase C inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione. In rat aortic rings U46619 caused TXA2 receptor-mediated contractions (EC50 of 28 +/- 2 nM) which were not attenuated by removal of extracellular Ca++ from the superfusion buffer. Together, these results suggest that agonist occupation of TXA2 receptors produces vascular smooth muscle contraction through initial activation of phospholipase C with production of 1,4,5-inositol phosphate, release of intracellular calcium stores and phosphorylation of myosin light chains associated with cellular acidification, presumably via activation of Ca++ ATPase.