Binding properties of C-truncated delta opioid receptors. (1/286)

AIM: To study the role of C-terminal delta opioid receptor involved in ligand binding affinity and selectivity. METHODS: The 31 amino acid residues of C-terminal truncated delta opioid receptors and the wild-type were expressed stably in Chinese hamster ovary (CHO) cells, respectively. Then the ligand binding properties of the products were studied by receptor binding assay. RESULTS: A typical mutated receptor clone CHO-T and a wild-type receptor clone CHO-W were obtained. The Kd values of [3H] diprenorphine (Dip) and [3H]leucine-2-alanine enkephalin (DADLE) bound to CHO-T were similar to CHO-W. Both the specific [3H]Dip bindings of CHO-T and CHO-W were strongly inhibited by delta selective agonists with similar Ki, but neither by mu nor kappa selective agonists. CONCLUSION: The C-terminal of the delta opioid receptor is not involved in the ligands binding affinity and selectivity.  (+info)

Activation of spinal opioid receptors contributes to hypotension after hemorrhage in conscious rats. (2/286)

Opioid receptors are activated during severe hemorrhage, resulting in sympathoinhibition and a profound fall in blood pressure. This study examined the location and subtypes of opioid receptors that might contribute to hypotension after hemorrhage. Intrathecal naloxone methiodide (100 nmol) abolished the fall in blood pressure after hemorrhage (1.5% of body wt; mean arterial pressure 122 +/- 8 mmHg after naloxone methiodide vs. 46 +/- 5 mmHg in controls, P < 0. 001). Intracisternal naloxone methiodide was less effective than intrathecal naloxone methiodide, whereas intravenous naloxone methiodide, which does not cross the blood-brain barrier, did not alter the fall in blood pressure after hemorrhage. These results demonstrate that spinal opioid receptors contribute to hypotension after hemorrhage but do not exclude supraspinal effects. In separate experiments, the subtype-specific opioid antagonists ICI-174864 (delta-antagonist), norbinaltorphimine (nor-BNI; kappa-antagonist), and H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; mu-antagonist) were each administered intrathecally to determine the minimum dose that would attenuate hypotension during severe hemorrhage. These antagonists were effective at similar doses (3 nmol for CTOP, 6 nmol for ICI-174864, and 10 nmol for nor-BNI), although the binding affinities of these three different agents for their target receptors varied >1600-fold. Comparisons of the minimum effective doses of these antagonists in relation to their binding affinities provides strong evidence for the participation of delta-receptors in mediating hypotension after hemorrhage. In contrast, the dose at which nor-BNI was effective suggests an effect at delta-receptors but not kappa-receptors. The efficacy of CTOP, albeit at a high dose, also suggests an effect at mu-receptors.  (+info)

Antagonistic effect of orphanin FQ on opioid analgesia in rat. (3/286)

AIM: To study the effect of orphanin FQ (OFQ), a newly discovered heptadecapeptide, on nociception and opioid analgesia. METHODS: The intracerebroventricular (i.c.v.) and intrathecal (i.t.h.) injections were used to give the drugs. The tail-flick model of rats were used to test the pain threshold. RESULTS: OFQ (i.c.v. or i.t.h.) 0.1 microgram had no effect on nociception but 0.5-10 micrograms induces hyper-reaction of rat to noxious electric stimulus; the decapeptide (OFQ1-10 i.c.v.), a fragment of the OFQ, did not affect the pain reaction of rats. Fentanyl (1 microgram, i.c.v. or i.t.h.), a selective mu-receptor agonist, DSLET (5 micrograms, i.c.v. or i.t.h.), a selective delta-receptor agonist, or U50488H (1 microgram, i.t.h.), a kappa-receptor agonist, induced an increase in pain threshold, when OFQ (0.1 or 1 microgram) was added together with one of them (except for the ith injection of DSLET), the increase of pain threshold was reduced obviously. CONCLUSION: OFQ induces hyperalgesia and antagonizes opioid analgesia mediated by mu- and delta-receptors in the brain and by mu- and kappa- but not delta-receptors in the spinal cord of rats.  (+info)

L-proline and L-pipecolate induce enkephalin-sensitive currents in human embryonic kidney 293 cells transfected with the high-affinity mammalian brain L-proline transporter. (4/286)

The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, particularly the Na gradient. In the nervous system, transporters clear synapses and help to replenish transmitters in nerve terminals. The localization of PROT to specific excitatory terminals in rat forebrain suggests a role for this carrier in excitatory transmission (). We investigated the voltage regulation and electrogenicity of this novel transporter, using human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and -40 mV, L-proline (PRO) and its six-member ring congener L-pipecolate (PIP) induced inward current. The current-voltage relationship and the variance of current fluctuations were similar for PRO- and PIP-induced current, and the ratio of induced variance to the mean current ranged from 20 to 60 fA. Des-Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of PROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current was absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated transport only when applied to the extracellular face of PROT. These data suggest that (1) PROT uptake is electrogenic, (2) individual transporter currents are voltage-independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally accessible pore.  (+info)

A parasympathetic ganglion innervating the harderian gland and lacrimal gland of the musk shrew (Suncus murinus): fluorescent tracing and immunohistochemical studies. (5/286)

A small ganglion, named the peri-trigeminal ganglion (PTG), was found in the ventromedial border of the rostral half of the trigeminal ganglion (TG) in the musk shrew (Suncus murinus). In frontal sections, the PTG was semicircular or elliptical in shape. Most of the neurons constituting this ganglion were round in shape and much smaller than those of the TG. The retrograde fluorescent tracer fluoro-gold was injected into various regions of the face in order to investigate innervation by the PTG neurons. When the tracer was injected subcutaneously around the external acoustic meatus and around the circumference of the orbit, a number of labeled neurons were seen not only in the TG but also in the PTG. After applying the tracer to the lacrimal gland (LG) and the harderian gland (HG), numerous labeled neurons were detected only in the PTG. A few labeled neurons were found in the PTG after injection into the palatoglossal arch. Immunohistochemically, most of the neurons constituting the PTG were positive for vasoactive intestinal polypeptide (VIP) antiserum. And a moderate number of somatostatin (SOM)-immunoreactive neurons and a small number of leucine-enkephalin (L-ENK)-immunoreactive neurons were detected. Numerous substance P-immunoreactive nerve fibers and varicosities were found in the PTG, and fewer L-ENK-, SOM- and VIP-immunoreactive fibers were observed. The present results suggest that the PTG is an autonomic ganglion that resembles in part the pterygopalatine ganglion in other species, and mainly innervates the HG and LG.  (+info)

Preproenkephalin is a Th2 cytokine but is not required for Th2 differentiation in vitro. (6/286)

Preproenkephalin (PPNK) mRNA expression has been detected in many cells of the immune system, including monocytes and lymphocytes. In the present paper, the expression of PPNK mRNA in purified CD4+ Th1 and Th2 lymphocyte subpopulations is investigated and correlated with the presence of the opioid neuropeptides leu- and met-enkephalin. We found that PPNK mRNA and met-enkephalin were present at higher levels in the Th2 cultures compared with the Th1 cultures. Lymphocytes from PPNK-deficient mice were then used to look at the role of PPNK in Th2 lymphocyte differentiation. Lymphocytes from these mice could be driven into a Th2 phenotype, suggesting that cultures containing IL-4 do not require PPNK for Th2 differentiation.  (+info)

Met5-enkephalin protects isolated adult rabbit cardiomyocytes via delta-opioid receptors. (7/286)

In rats and rabbits, endogenous opioid peptides participate in ischemic preconditioning. However, it is not known which endogenous opioid(s) can trigger cardioprotection. We examined preconditioning-induced and opioid-induced limitation of cell death in isolated, calcium-tolerant, adult rabbit cardiomyocytes. Cells were subjected to simulated ischemia by pelleting and normothermic hypoxic incubation. Preconditioning was elicited with 15 min of simulated ischemia followed by 15 min of resuspension and reoxygenation. All cells underwent 180 min of simulated ischemia. Cell death was assessed by trypan blue permeability. Morphine protected cells, as did preconditioning; naloxone blocked the preconditioning-induced protection. Exogenous Met5-enkephalin (ME) induced protection, but exogenous beta-endorphin did not. ME-induced protection was blocked by the delta-selective antagonist naltrindole. Additionally, two other proenkephalin products, Leu5-enkephalin and Met5-enkephalin-Arg-Phe, provided protection equipotent to ME. These data suggest that one or more proenkephalin products interact with delta-opioid receptors to endogenously trigger opioid-mediated protection.  (+info)

Antinociceptive effect and enzymatic degradation of endomorphin-1 in newborn rat spinal cord. (8/286)

Recently discovered endomorphin-1 and -2 are the first endogenous agonists selective for the mu-opioid receptor. We examined the antinociceptive effect and enzymatic degradation of endomorphin-1 in the newborn rat spinal cord. Endomorphin-1 inhibited the binding of [3H][D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin (DAMGO) to the membrane fraction of the newborn rat spinal cord as potently as DAMGO and morphine. Endomorphin-1 at 1-1,000 nM reduced the slow ventral root potential, which reflects noxious transmission in the isolated newborn rat spinal cord, concentration-dependently via the mu-opioid receptor. A similar effect was observed with endomorphin-2. The newborn rat spinal cord homogenate degraded endomorphin-1 in a 120-min incubation procedure, while it degraded [Leu5]enkephalin even in a 30-min incubation procedure. The degradation of endomorphin-1 was inhibited by actinonin but not by thiorphan. These results showed that in the newborn rat spinal cord, endomorphins had high affinity for the mu-opioid receptor and exerted mu-opioid-receptor-mediated inhibitory effects on noxious responses. Endomorphin-1 was degraded by peptidases, but slowly compared with [Leu5]enkephalin degradation, and the degrading enzymes were actinonin-sensitive peptidases.  (+info)