Ultraviolet A eye irradiation ameliorates colon carcinoma induced by azoxymethane and dextran sodium sulfate through ß-endorphin and methionine-enkephalin.

BACKGROUND: We previously reported that ultraviolet (UV) A eye irradiation reduces the ulcerative colitis induced by dextran sodium sulfate (DSS). This study examined the effects of UVA on colon carcinoma induced by azoxymethane (AOM) and DSS. METHODS: We irradiated the eyes of ICR mice with UVA at a dose of 110 kJ/m2 using an FL20SBLB-A lamp for the experimental period. RESULTS: In mice treated with these drugs, the symptom of colon carcinoma was reduced by UVA eye irradiation. The levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the blood were increased in AOM + DSS-treated mice; however, those levels were reduced by UVA eye irradiation. The expression of ß-endorphin, methionine-enkephalin (OGF), µ-opioid receptor, and opioid growth factor receptor (OGFR) of the colon was increased in the AOM + DSS-treated mice, and these levels were increased further following UVA eye irradiation. When ß-endorphin inhibitor was administered, the ameliorative effect of UVA eye irradiation was reduced, and the effect of eye irradiation disappeared entirely following the administration of naltrexone (inhibitor of both opioid receptor and OGFR). CONCLUSIONS: These results suggested that UVA eye irradiation exerts major effects on AOM + DSS-induced colon carcinoma.

Gestational choline supplementation normalized fetal alcohol-induced alterations in histone modifications, DNA methylation, and proopiomelanocortin (POMC) gene expression in ß-endorphin-producing POMC neurons of the hypothalamus.

BACKGROUND: Prenatal exposure to ethanol (EtOH) reduces the expression of hypothalamic proopiomelanocortin (POMC) gene, known to control various physiological functions including the organismal stress response. In this study, we determined whether the changes in POMC neuronal functions are associated with altered expressions of histone-modifying and DNA-methylating enzymes in POMC-producing neurons, because these enzymes are known to be involved in regulation of gene expression. In addition, we tested whether gestational choline supplementation prevents the adverse effects of EtOH on these neurons. METHODS: Pregnant rat dams were fed with alcohol-containing liquid diet or control diet during gestational days 7 and 21 with or without choline, and their male offspring rats were used during the adult period. Using double-immunohistochemistry, real-time reverse transcription polymerase chain reaction (RT-PCR) and methylation-specific RT-PCR, we determined protein and mRNA levels of histone-modifying and DNA-methylating enzymes and the changes in POMC gene methylation and expression in the hypothalamus of adult male offspring rats. Additionally, we measured the basal- and lipopolysaccharide (LPS)-induced corticosterone levels in plasma by enzyme-linked immunosorbent assay. RESULTS: Prenatal EtOH treatment suppressed hypothalamic levels of protein and mRNA of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, phosphorylated H3S10), and increased the repressive marks (H3K9me2, G9a, Setdb1), DNA-methylating enzyme (Dnmt1), and the methyl-CpG-binding protein (MeCP2). The treatment also elevated the level of POMC gene methylation, while it reduced levels of POMC mRNA and ß-EP and elevated corticosterone response to LPS. Gestational choline normalized the EtOH-altered protein and the mRNA levels of H3K4me3, Set7/9, H3K9me2, G9a, Setdb1, Dnmt1, and MeCP2. It also normalizes the changes in POMC gene methylation and gene expression, ß-EP production, and the corticosterone response to LPS. CONCLUSIONS: These data suggest that prenatal EtOH modulates histone and DNA methylation in POMC neurons that may be resulting in hypermethylation of POMC gene and reduction in POMC gene expression. Gestational choline supplementation prevents the adverse effects of EtOH on these neurons.

Suppression of acute morphine withdrawal syndrome by adenovirus-mediated ß-endorphin in rats.

BACKGROUND: Endogenous ß-endorphin (ß-EP) in the central nervous system (CNS) is decreased upon opioid addiction. The current study examined whether exogenous ß-EP, delivered using an adenoviral vector into the CNS could attenuate morphine withdrawal syndrome in rats. METHODS: The model of opioid-dependent rats was set up by receiving subcutaneous injection of morphine using an escalating regimen for 6days (5, 10, 20, 40, 50, 60mg/kg, three times/day). The adenovirus mediated ß-EP gene was constructed based on our previous work. The ilea of opioid-dependent rats were isolated and treated with the supernatant of Ad-NEP. The basic and naloxone-induced (4µm/l) contractions of dependent ilea were recorded. The Ad-NEP was injected into the left lateral ventricle of the addition rats. The expression of the ß-EP gene was verified by radioimmunoassay of the cerebrospinal fluid (CSF) and immunocytochemistry for ß-EP. Withdrawal syndrome was evaluated after intraperitoneal injection of naloxone. RESULTS: The contractions of dependent ilea were attenuated with supernatant containing ß-EP expressed by Ad-NEP. Injection of the Ad-NEP resulted in significant increases in ß-EP level in the CSF and ß-EP-positive neurons. Rats receiving adenovirus carrying the ß-EP gene had significantly less severe withdrawal symptoms upon naloxone challenge. CONCLUSIONS: Exogenous ß-EP mediated by adenovirus could attenuate withdrawal syndrome in morphine-dependent rats.

T lymphocytes containing ß-endorphin ameliorate mechanical hypersensitivity following nerve injury.

Neuropathic pain is a debilitating consequence of nerve injuries and is frequently resistant to classical therapies. T lymphocytes mediate adaptive immune responses and have been suggested to generate neuropathic pain. In contrast, in this study we investigated T cells as a source of opioidergic analgesic ß-endorphin for the control of augmented tactile sensitivity following neuropathy. We employed in vivo nociceptive (von Frey) testing, flow cytometry and immunofluorescence in wild-type and mice with severe combined immunodeficiency (SCID) subjected to a chronic constriction injury of the sciatic nerve. In wild-type mice, T lymphocytes constituted approximately 11% of all immune cells infiltrating the injury site, and they expressed ß-endorphin and receptors for corticotropin-releasing factor (CRF), an agent releasing opioids from leukocytes. CRF applied at the nerve injury site fully reversed neuropathy-induced mechanical hypersensitivity in wild-type animals. In SCID mice, T cells expressing ß-endorphin and CRF receptors were absent at the damaged nerve. Consequently, these animals had substantially reduced CRF-mediated antinociception. Importantly, the decreased antinociception was fully restored by transfer of wild-type mice-derived T lymphocytes in SCID mice. The re-established CRF antinociception could be reversed by co-injection of an antibody against ß-endorphin or an opioid receptor antagonist with limited access to the central nervous system. We propose that, in response to CRF stimulation, T lymphocytes accumulating at the injured nerves utilize ß-endorphin for activation of local neuronal opioid receptors to reduce neuropathy-induced mechanical hypersensitivity. Our findings reveal ß-endorphin-containing T cells as a crucial component of beneficial adaptive immune responses associated with painful peripheral nerve injuries.

beta-Endorphin regulates diverse functions of splenic phagocytes through different opioid receptors in freshwater fish Channa punctatus (Bloch): an in vitro study.

In this in vitro study, the role of beta-endorphin in the control of phagocytic and cytotoxic activities of fish splenic phagocytes was investigated. Further, the involvement of specific opioid receptor was explored. beta-Endorphin stimulated phagocytosis, whereas inhibited nitric oxide production as assessed by nitrite release. However, it had concentration-related biphasic effects on superoxide production, stimulatory at low and inhibitory at high concentration. Naltrexone, non-selective opioid receptor antagonist, antagonized the effect of beta-endorphin on phagocyte functions. Moreover, CTAP, selective mu-receptor antagonist, completely blocked the effect of beta-endorphin on phagocytosis and nitrite release. With regard to superoxide production, CTAP blocked the stimulatory effect of beta-endorphin at low concentration, while the inhibitory effect at high concentration was completely antagonized by selective delta-receptor antagonist, NTI. In conclusion, beta-endorphin acting via mu-receptor stimulated phagocytosis and inhibited nitric oxide production, while its biphasic effect on superoxide production seems to be mediated by mu- and delta-receptors.

Plasma met-enkephalin, beta-endorphin and leu-enkephalin levels in human hepatic encephalopathy.

To address the role of the opioid system in the pathogenesis of hepatic encephalopathy (HE) we measured plasma met-enkephalin, beta-endorphin and leu-enkephalin in patients with different grades of HE compared to control subjects and patients with cirrhosis. Plasma met-enkephalin levels were significantly higher in patients with cirrhosis and all grades of HE than controls. Plasma beta-endorphin levels were similar in the 3 groups. Plasma leu-enkephalin levels were significantly higher in HE grades II, III and IV than in controls, patients with cirrhosis and HE grade I patients. Our results support data on the involvement of met-enkephalin and leu-enkephalin in the pathogenesis of HE and provide a rationale for the use of opioid receptor antagonists in the treatment of HE.

Effects of opioid blockade with naltrexone and distraction on cold and ischemic pain in hypertension.

Essential hypertension is characterised by reduced pain sensitivity. Hypertensive hypoalgesia has been attributed to elevated endogenous opioids and/or increased activation of descending pain modulation systems. A double-blind placebo-controlled design compared the effects of naltrexone and placebo on cold and ischemic pain in unmedicated newly-diagnosed patients with essential hypertension. Patients performed a cold pressor task while resting and while performing a distracting secondary task. They also performed a forearm ischemia task while resting. Although the cold pressor and ischemia tasks elicited significant increases in pain and blood pressure, pain ratings and pressor responses did not differ between naltrexone and placebo. Cold pain was reduced by distraction compared to rest. The finding that opioid blockade with naltrexone did not moderate the pain and pressor responses to cold and ischemia suggests that pain and associated blood pressure responses are not modulated by opioids in hypertension. The finding that the distracting secondary task successfully reduced pain ratings suggests normal supraspinal pain modulation in essential hypertension.

Formalin pretreatment attenuates tail-flick inhibition induced by beta-endorphin administered intracerebroventricularly or intrathecally in mice.

We examined the effect of the subcutaneous (s.c.) pretreatment of formalin into both hind paws of mice on the antinociception induced by the intracerebroventricularly (i.c.v.) or intrathecally (i.t.) administration of beta-endorphin using the tail-flick test. Pretreatment with formalin (5%) for 5 h had no affect on the i.c.v. administered beta-endorphin-induced tail-flick response. However, pretreatment with formalin for 40 h attenuated the tail-flick inhibition induced by i.c.v. administered beta-endorphin. This antinociceptive tolerance to i.c.v. beta-endorphin continued up to 1 week, but to a lesser extent. Pretreatment with formalin for 5 and 40 h significantly reduced the i.t. beta-endorphin-induced inhibition of the tail-flick response, which continued up to 1 week. The s.c. formalin treatment increased the hypothalamic pro-opiomelanocortin (POMC) mRNA level at 2 h, but this returned to the basal level after 40 h. Our results suggest that the increase in the POMC mRNA level in the hypothalamus appears to be involved in the supraspinal or spinal beta-endorphin-induced antinociceptive tolerance in formalin-induced inflammatory pain.

No evidence for G-protein-coupled epsilon receptor in the brain of triple opioid receptor knockout mouse.

Pharmacological approaches have defined the epsilon receptor as a beta-endorphin-preferring opioid receptor, described in rat vas deferens and in brain of several species. Only three opioid receptors-mu, delta and kappa-have been cloned and the existence of this additional subtype as a distinct protein remains controversial. Recently, the mouse brain epsilon receptor was detected in a G protein activation assay, as mediating residual beta-endorphin activity following pharmacological blockade of mu, delta and kappa receptors. To clarify whether this site is independent from mu, delta and kappa receptors, we performed beta-endorphin-induced [(35)S]GTPgammaS binding using mice lacking these three receptors (triple knockout mice). We tested both pons-medulla and whole brain preparations. beta-Endorphin strongly stimulated [(35)S]GTPgammaS binding in wild-type membranes but had no detectable effect in membranes from triple knockout mice. We conclude that the brain epsilon site involves mu, delta and/or kappa receptors, possibly coupled to nonclassical G proteins.

A critical role for beta-endorphin in cocaine-seeking behavior.

Endogenous beta-endorphin levels in the brain are elevated in response to cocaine and are downstream of the mesolimbic dopaminergic system. However, beta-endorphin's direct involvement in cocaine reinforcement has not been demonstrated. In the present study, a single bilateral microinjection of anti-beta-endorphin antibodies (4 microg) to the nucleus accumbens during the maintenance phase of cocaine self-administration (1 mg/kg/infusion) significantly increased the number of active and inactive lever responses. The increase in lever responses is reminiscent of rat behavior during extinction of cocaine self-administration. Further, a cocaine dose-response demonstrates that the increased lever presses in anti-beta-endorphin antibody-injected rats was still present after substitution with a lower dose of cocaine. These findings support a critical role for beta-endorphin in the cocaine brain reward system.

Involvement of endogenous beta-endorphin in antinociception in the arcuate nucleus of hypothalamus in rats with inflammation.

Although exogenous administration of beta-endorphin to the arcuate nucleus of hypothalamus (ARC) had been shown to produce antinociception, the role of endogenous beta-endorphin of the ARC in nociceptive processing has not been studied directly. The aim of the present study was to investigate the effect of endogenous beta-endorphin in the ARC on nociception in rats with carrageenan-induced inflammation. The hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation was assessed by the hot-plate test and the Randall Selitto Test. Intra-ARC injection of naloxone had no significant influence on the HWL to thermal and mechanical stimulation in intact rats. The HWL decreased significantly after intra-ARC injection of 1 or 10 microg of naloxone in rats with inflammation, but not with 0.1 microg of naloxone. Furthermore, intra-ARC administration of the selective mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) decreased the nociceptive response latencies to both stimulation in a dose-dependent manner in rats with inflammation, while intra-ARC administration of the selective delta-opioid receptor antagonist naltrindole or the selective kappa-opioid receptor antagonist nor-binaltorphimine (nor-BNI) showed no influences on the nociceptive response latency. The antiserum against beta-endorphin, administered to the ARC, also dose-dependently reduced the HWL in rats with inflammation. The results indicate that endogenous beta-endorphin in the ARC plays an important role in the endogenous antinociceptive system in rats with inflammation, and that its effect is predominantly mediated by the mu-opioid receptor.

Pivotal roles of alpha-melanocyte-stimulating hormone and the melanocortin 4 receptor in leptin stimulation of prolactin secretion in rats.

Leptin, the obese gene product, was reported to stimulate prolactin (PRL) secretion, but the neuroendocrine mechanism underlying this hormonal response is largely unknown. Thus, in this study we examined the involvement of several important PRL regulators in the leptin-induced PRL secretion in male rats. Compared with the values in normally fed rats, food deprivation for 3 days significantly decreased both PRL and leptin levels in the plasma. These changes were reverted to normal by a 3-day constant infusion of 75 microg/kg/day of leptin to the fasted rats, while 225 microg/kg/day of leptin further elevated both PRL and leptin levels. These four groups of animals were used for the following experiments. Results of dopamine and serotonin turnover studies in the brain and the pituitary indicated that neither of these biogenic amines plays a primary role in mediating leptin's effects on PRL. Repeated intracerebroventricular injections over 72 h of neutralizing antibodies against vasoactive intestinal peptide, PRL-releasing peptide, or beta-endorphin, did not significantly suppress the leptin actions. However, both the blockade of the melanocortin (MC) 4 receptor (R) and the immunoquenching of brain alpha-melanocyte-stimulating hormone (alpha-MSH) completely abolished the leptin-induced PRL release, and the stimulation of the MC4-R, but not the MC3-R, significantly elevated PRL levels in the fasted rats. These results suggest that alpha-MSH, a cleaved peptide from pro-opiomelanocortin of which synthesis is stimulated by leptin, may be the pivotal neuropeptide in the brain mediating the leptin's stimulatory influence on PRL secretion. It was also suggested that the MC4-R may be the primary subtype of the MC-Rs mediating this action of alpha-MSH.

Antagonistic property of buprenorphine for putative epsilon-opioid receptor-mediated G-protein activation by beta-endorphin in pons/medulla of the mu-opioid receptor knockout mouse.

beta-Endorphin is a non-selective opioid peptide which binds mu-, delta- and putative epsilon (beta-endorphin-sensitive non-mu-, non-delta- and non-kappa(1)-)-opioid receptors. We have previously reported that beta-endorphin-produced G-protein activation is mediated by the stimulation of both mu- and putative epsilon-opioid receptors. The present study was designed to further characterize this putative epsilon-opioid receptor-mediated G-protein activation in the pons/medulla membrane obtained from mice lacking mu-opioid receptor, using a guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS)-binding assay. beta-Endorphin and the mu-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) increased the [(35)S]GTPgammaS binding in a concentration-dependent manner (0.001-10 microM), and at 10 microM beta-endorphin and DAMGO produced approximately 250 and 120% increases of [(35)S]GTPgammaS binding in the pons/medulla membrane obtained from wild-type mice, respectively. In the pons/medulla membrane obtained from mu-opioid receptor knockout mice, beta-endorphin-stimulated [(35)S]GTPgammaS binding was only partially attenuated and a more than 100% increase by 10 microM beta-endorphin still remained, while DAMGO failed to produce any increase in [(35)S]GTPgammaS binding. The residual increase in [(35)S]GTPgammaS binding by 10 microM beta-endorphin in mu-opioid receptor knockout mice was partially but significantly attenuated by the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27), but not by the delta-opioid receptor antagonist naltrindole or the kappa(1)-receptor antagonist norbinaltorphimine. Furthermore, buprenorphine significantly attenuated the residual increase in [(35)S]GTPgammaS binding by 10 microM beta-endorphin in mu-opioid receptor knockout mice. The present results indicate that beta-endorphin activates G-protein by stimulation of putative epsilon-opioid receptors in the condition lacking the mu-opioid receptor, and buprenorphine acts as an antagonist for putative epsilon-opioid receptors in this condition.

Plasma glucose-lowering effect of beta-endorphin in streptozotocin-induced diabetic rats.

The effect of beta-endorphin on plasma glucose levels was investigated in streptozotocin-induced diabetic rats (STZ-diabetic rats). A dose-dependent lowering of plasma glucose was observed in the fasting STZ-diabetic rat fifteen minutes after intravenous injection of beta-endorphin. The plasma glucose-lowering effect of beta-endorphin was abolished by pretreatment with naloxone or naloxonazine at doses sufficient to block opioid mu-receptors. Also, unlike wild-type diabetic mice, beta-endorphin failed to induce its plasma glucose-lowering effect in the opioid mu-receptor knock-out diabetic mice. In isolated soleus muscle, beta-endorphin enhanced the uptake of radioactive glucose in a concentration-dependent manner. Stimulatory effects of beta-endorphin on glycogen synthesis were also seen in hepatocytes isolated from STZ-diabetic rats. The blockade of these actions by naloxone and naloxonazine indicated the mediation of opioid mu-receptors. In the presence of U73312, the specific inhibitor of phospholipase C (PLC), the uptake of radioactive glucose into isolated soleus muscle induced by beta-endorphin was reduced in a concentration-dependent manner, but it was not affected by U73343, the negative control of U73312. Moreover, chelerythrine and GF 109203X diminished the stimulatory action of beta-endorphin on the uptake of radioactive glucose at a concentration sufficient to inhibit protein kinase C (PKC). The data obtained suggest that activating opioid mu-receptors by beta-endorphin may increase glucose utilization in peripheral tissues via the PLC-PKC pathway to lower plasma glucose in diabetic rats lacking insulin.

Exercise under hot conditions: a major threat to the immune response?

It seems likely that the disturbances of immune response induced by prolonged competitive exercise are exacerbated if athletes also face the stress of hot environmental conditions. We have investigated this question by manipulating the exercise-induced increases of body temperature in a climatic chamber and by submersion of exercisers in a large water-bath. Hot conditions increase the stress of a given bout of exercise, as assessed by personal perceptions, objective (heart rate variability) measures of autonomic nerve balance, and the secretion of "stress" hormones, with a parallel increase in effects upon critical lymphocyte subsets. Changes in the immune response show substantial correlations with plasma concentrations not only of epinephrine (which modulates the adhesiveness of peripherally sequestered lymphocytes), but also with norepinephrine. The latter hormone may mobilize leukocytes from the spleen and lymph glands, or it may act by increasing cardiac output and thus intravascular shear forces. Given the cumulative impact of various environmental stressors upon the immune system, every effort should be made to minimize the athlete's exposure to stresses other than the exercise to be performed. In some circumstances, the use of medications to reduce the overall stress response may also be warranted.

The role of spinal cholecystokinin in chronic pain states.

It is well established that cholecystokinin (CCK) reduces the antinociceptive effect of opioids. The level of CCK and CCK receptors, as well as CKK release, exhibits considerable plasticity after nerve injury and inflammation, conditions known to be associated with chronic pain. Such altered CCK release coupled in some situation with changes in CCK receptor levels may underlie the clinical phenomenon of varying opioid sensitivity in different clinical pain conditions. In particular, neuropathic pain after injury to the peripheral and central nervous system does not respond well to opioids, which is likely to be caused by increased activity in the endogenous CCK system. CCK receptor antagonists may thus be useful as analgesics in combination with opioids to treat neuropathic pain.

Neonatal monosodium glutamate treatment abolishes both delta opioid receptor-induced and alpha-2 adrenoceptor-mediated gastroprotection in the lower brainstem in rats.

Neonatal monosodium glutamate treatment reduced immunoreactive beta-endorphin content in the mediobasal hypothalamus by 50% in adult, male Wistar rats as compared to hypertonic saline-treated littermates; there was also a moderate (approx. 25%) reduction in the rostral part of the nucleus of the solitary tract. In sham-treated adults the intracisternally injected alpha-2 adenoceptor stimulant clonidine (0.47 nmol/rat) and the delta opioid receptor type agonist (D-Ala(2), D-Leu(5))-enkephalin (0.8 nmol/rat) reduced acidified ethanol-induced mucosal lesions in the stomach by 84.1 and 77.5%, respectively, whereas the same doses were completely ineffective in rats treated neonatally by monosodium glutamate. The data taken together with the results of previous studies with the same substances in rats with retroarcuate knife cuts suggest that neuronal damage in the nucleus of the solitary tract region rather than in the arcuate nucleus is responsible for the changes seen in the pharmacological responsiveness.

Antagonism of nitrous oxide antinociception in mice by intrathecally administered antisera to endogenous opioid peptides.

Previously it was demonstrated that nitrous oxide antinociception in the mouse abdominal constriction test is mediated by kappa-opioid receptors. Since nitrous oxide is thought to cause the neuronal release of endogenous opioid peptide to stimulate opioid receptors, this study was designed to identify the opioid peptides involved, especially in the spinal cord, by determining whether nitrous oxide antinociception can be differentially inhibited by intrathecally (i. t.) administered antisera to different opioid peptides. Male NIH Swiss mice were pretreated i.t. with rabbit antisera to opioid peptides then exposed 24 h later to one of three different concentrations of nitrous oxide in oxygen. Dose-response curves constructed from the data indicated that the antinociceptive effect of nitrous oxide was significantly antagonized by antisera to various dynorphins (DYNs) and methionine-enkephalin (ME), but not by antiserum to beta-endorphin (beta-EP). The AD(50) values for nitrous oxide antinociception were significantly elevated by antisera to DYNs and ME but not beta-EP. These findings of this study support the hypothesis that nitrous oxide antinociception in the mouse abdominal constriction test involves the neuronal release of DYN and ME in the spinal cord.

Glycyl-L-glutamine injected centrally suppresses alcohol drinking in P rats.

Recent reports show that central beta-endorphin (1-31) injection augments the volitional intake of alcohol. Correspondingly, alcohol drinking stimulates beta-endorphin (1-31) release from the hypothalamus of the rat. Glycyl-l-glutamine (Gly-Gln) is produced in beta-endorphin-containing neurons and is co-released with beta-endorphin(1-31) and other processing products. Because Gly-Gln is apparently an endogenous antagonist of beta-endorphin(1-31) in several systems, the present study was designed to investigate the hypothesis that Gly-Gln injected i.c.v. would alter voluntary alcohol drinking in the genetic, high-alcohol-preferring P rat. After a guide tube was implanted stereotaxically above the lateral cerebral ventricle, the rats were offered 3-30% alcohol over 10 days, and then given their maximally preferred concentration of alcohol in the presence of water for the remainder of the experiment. Gly-Gln or artificial cerebrospinal fluid (CSF) vehicle then was injected i.c.v. in a dose of 10 or 100 nmol for 3 consecutive days, which was followed by a 7-day postinjection interval. Gly-Gln suppressed significantly the intakes of alcohol in terms of both g/kg and proportion to total fluid. During the postinjection days, alcohol drinking continued to be suppressed, whereas neither the daily intakes of food or water nor the body weights of the rats were changed. The present results are consistent with the concept of a functional antagonism by Gly-Gln of the role of beta-endorphin(1-31) in mediating certain central functions. These results demonstrate that alcohol consumption is suppressed by the direct intracerebral application of this unique peptide.

Liposomal delivery of a 30-mer antisense oligodeoxynucleotide to inhibit proopiomelanocortin expression.

An oligodeoxynucleic sequence of 30 bases (30-mer ODN), complementary to a region of beta-endorphin mRNA, was synthesized to have an antisense effect with regard to the expression of this oligopeptide. Following the solid-phase synthesis of the oligodeoxynucleotide, the 30-mer ODN was encapsulated within liposomes to provide a higher resistance against DNases and an improved entrance into cells. The most suitable liposome formulation as a 30-mer ODN carrier consisted of small unilamellar vesicles (50 nm) with an encapsulation capacity of 4.76 microL/micromol. The liposomal formulations containing dipalmitoyl-DL-alpha-phosphatidyl-L-serine presented fusogenic properties, which are of great importance for the delivery of antisense compounds. The antisense activity of 30-mer ODN-loaded liposomes was evaluated by the determination of beta-endorphin levels in AtT-20 cells. The free 30-mer ODN did not provide any lowering of the beta-endorphin production, whereas the liposomally entrapped compound elicited a concentration-dependent inhibition. The inhibition was determined by a sequence-specific binding of the 30-mer ODN with the target mRNA.