Presynaptic modulation by noradrenaline and an opioid of the substance P-induced release of [3H]acetylcholine from the myenteric plexus

Substance P (7.5-750 nM) applied in superfusion dose-dependently released 3H from isolated strips of myenteric plexus-longitudinal muscle of the guinea-pig ileum loaded with [3H]choline. Separation of the [3H]acetylcholine and [3H]choline components of the released radioactivity revealed that in response to substance P (SP) administration only the release of [3H]acetylcholine increased above resting level. A slowly developing tachyphylaxis to the effect of SP was observed. Evidence has been obtained that the slow tachyphylaxis developed to the acetylcholine-releasing effect of SP was not due to the exhaustion of releasable acetylcholine pool. Release of acetylcholine by 150 nM SP was completely prevented by tetrodotoxin or in a Ca2+-free medium and greatly reduced in the presence of noradrenaline or the opioid receptor agonist (D-Met2,Pro5)-enkephalinamide. The effect of noradrenaline and the opioid peptide was apparently prevented by yohimbine and naloxone, respectively.     

Kinetics of four 11C-labelled enkephalin peptides in the brain, pituitary and plasma of rhesus monkeys

The kinetics of four 11C-labelled enkephalin peptides: Tyr-Gly-Gly-Phe-Met (Met-enkephalin), Tyr-D-Met-Gly-Phe-Pro-NH2 [D-Met2,Pro5)-enkephalinamide), Tyr-D-Ala-Gly-Phe-Met-NH2 (DALA) and Tyr-D-Ala-D-Ala-Phe-Met-NH2 (TAAFM) all labelled at the methyl group of methionine was studied in the Rhesus monkey. After intravenous administration, the regional kinetics in the head, lungs, liver and kidneys were followed by means of positron emission tomography (PET). The total radioactivity in blood and urine was measured and the composition of 11C-labelled peptide fragments in plasma in vivo and in vitro was analysed by liquid chromatography. With PET, an increased radioactivity was observed in the brain and pituitary over the 60-90 min investigation period after i.v. injection of the peptides. The highest radioactivities were noted for Met-enkephalin, followed by DALA and D-Met2, Pro5-enkephalinamide, while very low radioactivities were found for TAAFM. The uptake of Met-enkephalin- and DALA-derived radioactivity was of the same order as has previously been shown for morphine in the brain and considerably higher than that of D-Met2,Pro5-enkephalinamide and TAAFM, respectively. A large fraction of the brain radioactivity derived from Met-enkephalin and DALA probably emanated from [11C]methionine as indicated by plasma and urine analysis. Met-Enkephalin was rapidly eliminated from plasma in vitro with an half-life of less than two minutes, whereas DALA was stable suggesting clearance by other tissues than plasma. In conclusion, both Met-enkephalin and DALA, were rapidly hydrolyzed in vivo to [11C]methionine. [11C]Methionine was probably taken up in the brain, as the radioactivity increased with time in different brain regions as measured with PET.D-Met2,Pro5-Enkephalinamide and TAAFM were virtually stable in vivo and at least part of the radioactivity observed in the brain may have represented the intact peptide.     

Effect of estradiol on expression and activation of Akt protein in rat hypothalamus exposed to chronic [D-Met2, Pro5]-enkephalinamide treatment

Adult ovariectomized rats were implanted with [D-Met2, Pro5]-enkephalinamide (ENK)-containing osmotic minipumps. Two hours prior to sacrifice, some animals were treated with estradiol-17beta (E2) at a dose 10 microg/100 g bodyweight (BW). Expression and activation of Akt proteins, nuclear [3H]estradiol binding, and the expression of estrogen receptor alpha (ERalpha) and beta (ERbeta) and of progesterone receptor (PR) were investigated. Estradiol increased the level of activated Akt protein (pAkt473) in the hypothalamus by 52 +/- 11% in comparison to the vehicle-treated controls. No such effect of E2 was observed 24 and 48 h after ENK implantation. This effect of ENK was abolished by concomitant treatment with naloxone. Time-dependent changes in nuclear [3H]estradiol binding and the expression of estrogen and progesterone receptors were also detected in the hypothalamus of ENK-implanted and E2-treated rats. At 24-48 h following ENK implantation, expression of ERalpha and high affinity [3H]estradiol binding decreased. At this time point, the PR level was also reduced, while the ERbeta level was augmented. In conclusion, these results suggest that the stimulatory effects of E2 on the expression and activation of Akt protein and the expression of ERalpha and PR are negatively regulated in rat hypothalamus exposed to chronic ENK treatment.     

Developmental changes in the inhibition of cultured rat uterine cell proliferation by opioid peptides

Opioid peptides are negative regulators of cell proliferation in several organs including the uterus. In the present study, the ontogeny of the direct inhibitory action of opioid peptides on the proliferation of cultured rat uterine cells was investigated. Uteri of 7, 14, 21, 28, 35 and 60-day-old rats were removed in a sterile way. Tissue blocks were dispersed by limited digestions with trypsin and collagenase. Cells were cultured in enriched Dulbecco's modified Eagle's medium (DMEM). Treatments were present during the entire culture period. Cell densities of the monolayers were determined by counting the cells following trypsinization and trypan blue exclusion. Rat uterine mixed cell cultures grew to confluence within 10 days. The average population doubling time gradually increased with the age of animals. Epidermal growth factor (EGF) increased cell densities of cultures from all age groups. The oestradiol (E2)-responsiveness appeared at 21 days of age. The effect of [D-Met2-Pro5]-enkephalinamide (ENK) was biphasic. ENK and [Met5]-enkephalin (OGF) decreased cell densities of both unstimulated and EGF-stimulated cultures from 7-day-old rats to the same extent. ENK failed to act in 14-day-old animals. From 21 days of age on, the E2- or EGF-stimulated proliferation was inhibited only by ENK and DAMGO, while 30 nm DPDPE, Dynorhin-A, OGF, [Leu5]-enkephalin, beta-endorphin, and morphiceptin were ineffective. The half-inhibitory concentration of ENK was 0.3 nm. The effects of ENK were prevented by concomitant treatment with naloxone. Our novel data demonstrate two different phases of the inhibitory action of opioid peptides on rat uterine cell proliferation during ontogeny with an insensitive interval in between.     

A behavioral role for enkephalins in regulating locomotor activity in the insect Leucophaea maderae: evidence for high affinity kappa-like opioid binding sites

D'-Ala-2 met-5-enkephalinamide application to the cerebral ganglia of Leucophaea maderae results in a decrease in locomotor activity. The opiate antagonist, naloxone, can block this effect as well as the depressant effect of morphine on locomotor activity. D-Ala-2,leu-5-enkephalinamide and dynorphin enhance locomotor activity following their topical application to the cerebral ganglia. This effect also can be antagonized by concomitant naloxone treatment. Benzomorphans were the most potent ligands tested in their ability to displace [3H]D'-ala-2,met-5-enkephalinamide whereas mu and delta ligands were by comparison less potent. These results suggest the presence of kappa-like opioid receptors in Leucophaea cerebral ganglia. The kappa ligands also are potent in enhancing locomotor activity in addition to being weakly antagonized by naloxone. Again, these results indicate the presence of multiple-opiate receptor types in invertebrates.     

Effect of naloxone and D-Met2-Pro5-enkephalinamide treatment on the DNA synthesis in the developing rat brain

Effects of a single dose of naloxone and of D-Met²-Pro⁵-enkephalinamide on the DNA synthesis in the forebrain, hypothalamus and cerebellum of 11 day old female rats were studied. As an index of DNA synthesis the rate of incorporation of ³H-thymidine into DNA was measured 30 min after a sc. injection of 40 μCi/100 g b.w.. A time dependent effect of naloxone administration on cerebral DNA synthesis was observed. In the forebrain at 1 and 3 hrs after naloxone injection an increased rate of ³H-thy-midine incorporation into DNA was found followed by a marked decrease at 9 and 12 hrs. The effect in the hypothalamus was similar but the initial increase at 1 hr was absent. On cerebellar DNA synthesis naloxone had no effect. The administration of D-Met²-Pro⁵-enkephalinamide resulted in a marked reduction in the labelling of cerebral and hypothalamic DNA between 1 to 12 hrs. Except a decrease at 1 hr no effect was found in the cerebellum.     

Modulation of dopamine-induced hypermotility following injection of various opioids into the nucleus accumbens

The aim of the present work was to obtain some data on the eventual role of nucleus accumbens in the antidopamine action of some opioids. Classical neuroleptics are known to inhibit the dopamine-elicited hypermotility when injecting them into the nucleus accumbens of rats pretreated with MAO inhibitors. In the present study the effects of some opioids have been examined in this model. The opioids examined were morphine, a mu-selective classical opiate, D-Ala2, Nle5-enkephalin sulphonic acid (ES), a delta selective opioid peptide and D-Met2, Pro5-enkephalinamide (EA), a non-selective opioid peptide. Haloperidol and chlorpromazine have been used for comparison. EA and morphine, especially the former, potently antagonized the dopamine-induced hyperactivity, similarly to haloperidol and chlorpromazine. ES exerted biphasic effect, the initial inhibition was followed by potentiation of the dopamine-elicited excitation. Thus the order of potency was: EA greater than haloperidol approximately equal to morphine greater than chlorpromazine greater than EA. The data indicate that the antidopamine action of opioids might be mediated, at least in part, by mu-receptors in the nucleus accumbens.     

Modulation of luteinizing hormone and follicle-stimulating hormone in circulation by interactions between endogenous opioids and oestradiol during the peripubertal period of heifers.

The aim of this study was to determine whether the decline in oestradiol inhibition of circulating luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the peripubertal period of heifers is associated with a change in opioid modulation of LH and FSH secretion. Opioid inhibition of LH secretion was determined by response to administration of the opioid antagonist naloxone. Prepubertal heifers (403 days old) were left as intact controls, ovariectomized or ovariectomized and chronically administered oestradiol. Control heifers were used to determine time of puberty. Three weeks after ovariectomy, four doses of naloxone (0.13-0.75 mg kg-1 body weight) or saline were administered to heifers in the treatment groups in a latin square design (one dose per day). Blood samples were collected at intervals of 10 min for 2 h before and 2 h after administration of naloxone. This procedure was repeated four times at intervals of 3 weeks during the time intact control heifers were attaining puberty. All doses of naloxone induced a similar increase in concentration of serum LH within a bleeding period. During the initial bleeding period (before puberty in control heifers), administration of naloxone induced an increase in LH concentration, but the response was greater for heifers in the ovariectomized and oestradiol treated than in the ovariectomized group. At the end of the study when control heifers had attained puberty (high concentrations of progesterone indicated corpus luteum function), only heifers in the ovariectomized and oestradiol treated group responded to naloxone. Opioid inhibition of LH appeared to decline in heifers during the time control heifers were attaining puberty. Heifers in the ovariectomized group responded to naloxone at the time of administration with an increase in FSH, but FSH did not respond to naloxone at any other time. Administration of naloxone did not alter secretion of FSH in ovariectomized heifers. These results suggest that opioid neuropeptides and oestradiol are involved in regulating circulating concentrations of LH and possibly FSH during the peripubertal period. Opioid inhibition of gonadotrophin secretion appeared to decline during the peripubertal period but was still present in ovariectomized heifers treated with oestradiol after the time when age-matched control heifers had attained puberty. We conclude that opioid inhibition is important in regulating LH and FSH in circulation in heifers during the peripubertal period. However, opioids continue to be involved in regulation of circulating concentrations of LH after puberty.     

[D-Ala, Met]-Enkephalinamide: CNS-mediated inhibition of prostaglandin-stimulated intestinal fluid and ion transport in the rat

The opioid peptide [D-Ala², Met⁵]-enkephalinamide (DAMA), a non-selective opioid agonist, has previously been shown to inhibit cholera toxin-induced fluid accumulation in the rat and dog small intestine after its intracerebroventricular (ICV) administration. In the present study, we examined the time course of the antisecretory/proabsorptive effects of ICV DAMA on net fluid and ion transport across the rat jejunum in situ during intravenous prostaglandin E₁ (PGE) infusion. Net water and NaCl absorption were measured using a standard dilution marker technique in a 15–20 cm segment of proximal jejunum in urethaneanesthetized Sprague-Dawley rats. Infusion of PGE (5 μg/kg-min) over a 2 hr period produced a decrease in fluid and ion absorption that plateaued to a steady-state within 60 min. DAMA (1 and 3 μg/rat) administered by ICV bolus 60 min after the start of PGE infusion inhibited significantly PGE-induced decreases in water and chloride absorption relative to saline-treated controls. These dose-related peptide effects were expressed 15 min after DAMA treatment and were approximately 30 min in duration; they were antagonized by naloxone (1 mg/kg, IV) given at the time of DAMA injection. These results indicate that low concentrations of DAMA administered into the central nervous system rapidly and effectively inhibit changes in intestinal transport induced by a blood-borne secretagogue through an interaction with opiate receptors.     

The opioid growth factor, [Met5]-enkephalin, inhibits DNA synthesis during recornification of mouse tail skin

Opioid peptides serve as tonically active negative growth regulators in renewing and regenerating epithelia. To examine the involvement of opioids in renewal of the stratum corneum after tape stripping of tail skin, C57BL/6 J mice were given systemic injections of the potent opioid antagonist, naltrexone (NTX, 20 mg/kg i.p.) following injury. Blockade of opioidreceptor interaction by NTX for 4 h resulted in an elevation of 36–;66% in basal cell DNA synthesis measured 24 h after injury. Injection of the endogenous opioid peptide, [Met⁵]-enkephalin (OGF, 10 mg/kg i.p.) 4 h before termination, suppressed radiolabelled thymidine incorporation in the basal cell layer by 37–46%at 24 h after wounding. The magnitude of the effects on DNA synthesis of OGF, but not NTX, depended on the timing of administration with respect to injury. OGF maximally depressed basal cell labelling (72%) when given 16 h after tape stripping. Concomitant administration of naloxone (10 mg/kg) with OGF blocked the inhibition of DNA synthesis; naloxone alone at the dosage utilized had no effect on cell labelling. Both OGF and its receptor, OGFr, were detected by immunocytochemistry in the basal and suprabasal cell layers, but not the cornified layer of tape stripped and uninjured tail skin. These results indicate: (a) a native opioid peptide and its receptor are expressed in epidermal cells of injured and uninjured mouse tail skin; (b) removal of the stratum corneum by tape stripping does not disrupt the function of the endogenous opioid growth system; (c) the proliferative response to wounding of the tail is tonically inhibited by the receptor-mediated action of an endogenous opioid peptide; and (d) DNA synthesis by basal cells can be elevated by disrupting opioid peptidereceptor interactions.     

Intratesticular injection of [D-Met2-Pro5]enkephalinamide suppresses testosterone secretion of the testis of immature rat

The possible role of enkephalin in the local control of testicular function was studied in neonatal rats. 5- and 10-day old hemicastrated rats were treated intratesticularly with an enkephalin analog [D-Met2-Pro5]enkephalinamide. In 5-day-old rats local injection of different doses (0.1-0.3 micrograms/testis) of the peptide suppressed basal testosterone secretion in vitro in a dose-dependent manner 2 h posttreatment. Intratesticular administration of naloxone prior to enkephalin treatment prevented the decrease in basal testosterone production induced by the opioid agonist. In 10-day-old animals intratesticular injection of 1.0 and 3.0 micrograms/testis of enkephalinamide reduced serum testosterone concentration and basal testosterone secretion in vitro. Systemic injection of the peptide produced no change in steroidogenesis. These results suggest that enkephalins might be among the intratesticular factors regulating Leydig cell functions.     

Effect of met-enkephalin and (d-Met2,Pro5)-enkephalinamide on the adenylate cyclase activity of rat brain

Among opiatelike peptides, stimulatory as well as inhibitory effects are encountered on adenylate cyclase activity. These actions are dependent not only on the investigated brain region but also on the applied peptide. Met-enkephalin stimulates adenylate cyclase activity in the rat brain stem (D-Met2, Pro5)-enkephalinamide and beta-endorphin inhibited it, whereas all three peptides inhibited the activity of cortex. Naloxone antagonized the effects of the applied peptides in the presence of sodium chloride.     

Effects of 9-ene-tetrahydrocannabinol on uterine estrogenicity in the mouse.

Several early (Phase I) and late (Phase II) estrogenic effects of 9-ene-tetrahydrocannabinol (THC) were examined in the adult mouse uterus. An injection of THC (2.5 or 10 mg/kg body wt) in ovariectomized mice neither stimulated uterine water imbibition or accumulation of [125I]bovine serum albumin (Phase I responses) at 6 h, nor antagonized these Phase I responses elicited by estradiol-17 beta (E2). With respect to Phase II responses, although single injections of THC (2.5, 5.0 and 10 mg/kg body wt) alone were ineffective in influencing uterine weight at 24 h or incorporation of [3H]thymidine at 18 h, this drug interfered with these responses elicited by E2 in a dose-dependent manner. In contrast, an injection of THC in progesterone (P4)-primed ovariectomized mice modestly enhanced (61%) uterine incorporation of [3H]thymidine. However, E2-stimulated uterine thymidine incorporation in P4-primed ovariectomized mice was antagonized by THC treatment. Effects of THC on blastocyst implantation were examined. Single or multiple injections of various doses of THC neither induced implantation in P4-primed delayed implanting mice, nor interfered with E2-induced implantation. Furthermore, daily injections of THC (10 mg/kg body wt) during the peri-implantation period had no apparent adverse effects on implantation, or on experimentally induced decidualization (deciduomata). The data suggest that THC is neither pro- nor antiestrogenic with respect to Phase I responses. However as regards Phase II responses, THC is modestly pro-estrogenic in the P4-treated uterus, but is anti-estrogenic in the presence of E2. These estrogen agonistic/antagonistic effects of THC on uterine Phase II responses do not adversely affect the process of implantation and decidualization.     

Dermorphin gene sequence peptide with high affinity and selectivity for delta-opioid receptors

Skin of the frog Phyllomedusa sauvagei contains a cDNA sequence that codes for the selective mu-receptor peptide dermorphin and a new heptapeptide we have designated as dermorphin gene-associated peptide (DGAP). Investigation of the opioid receptor binding characteristics of synthetic DGAP and [D-Met2]DGAP revealed that the latter peptide had high affinity and selectivity for delta-type opioid receptors in rat brain synaptosomes. The IC50 values for DGAP on mu- and delta-receptors were only 28 microM and 670 nM, respectively, while that for [D-Met2]DGAP was 0.80 nM for delta-receptors and greater than 1 microM for mu-receptors yielding a very high delta selectivity ratio (SR) of 1345. In comparison, the SR values for [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Pen2,5]enkephalin, ligands which are considered to be specific for delta-receptors, were 20, 42, and 301, respectively. Dermorphin, which contains a D-Ala2 residue and is a selective mu-receptor ligand (Lazarus, L.H., Guglietta, A., Wilson, W.E., Irons, B.J., and de Castiglione, R. (1989) J. Biol. Chem. 264, 354-362), exhibits a SR of 0.0055 similar to that for the conventional mu-agonist [D-Ala2,NMePhe4,Gly-ol]enkephalin (0.0040). This finding that frog skin cDNA contains the information to code for dermorphin and DGAP, or the presumed [D-Met2]DGAP molecule, which are among the most selective high affinity opioid ligands described for mu- and delta-receptors, may permit new insight into the design of future opioid receptor agonists and antagonists.     

Thymidine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules

Rats accustomed to eating during the first 8h of a daily 12h dark period re-established about 80% of intact liver weight, protein and DNA within 4 days following partial hepatectomy; further increases were not observed. Liver thymidine kinase activity and thymidine incorporation into liver DNA exhibited marked daily oscillations during liver regeneration. Maximum values were observed near the end of the dark period both in intact growing rats and in rats partially hepatectomized 2h before the end of the dark period. The time of day of surgery affected thymidine kinase activity and thymidine incorporation into DNA at specific times following partial hepatectomy. This seriously affects the interpretation of reports of experiments where the time of day of killing has been held constant and time of surgery varied. Highly significant correlation coefficients were observed for thymidine incorporation before killing versus thymidine kinase activity at time of killing and for thymidine versus orotic acid incorporation into DNA of livers from rats partially hepatectomized 2h before the end of the dark period and killed at 12h intervals. Thymidylate phosphatase activity returned to the normal amount at a rate similar to that for liver protein. Thymidylate phosphatase did not affect the validity of the thymidine kinase assay. The relationship of [(14)C]orotic acid to [(3)H]thymidine incorporation into liver DNA varied with the time of day, with the age of the rat and during the regeneration of the liver.     

On the mechanism of opioid-oestradiol interactions

Characteristics of opioid binding and possible relationships between oestradiol and opioid binding sites were studied in rat oestrogen sensitive tissues(uterus, preoptic area-anterior hypothalamus, median eminence-basal hypothalamus). Naloxone (Nal) and oestradiol (Oe) bindings were assessed by in vitro saturation analyses. In 800 g supernatants of both uterine and hypothalamic tissues homogenates high affinity (Kd: 2-4 X 10(-9) M) and low capacity [3H]Nal binding sites were found. These binding sites were sedimented from 800 g supernatant by further centrifugation at 10(5) g for 1 h. In competition studies [3H]Nal binding was completely prevented by morphine, while met-enkephalin and leu-enkephalin caused only a partial inhibition. [3H]Nal binding was increased by ovariectomy and decreased by Oe treatment (10 micrograms/100 g b.wt) in both tissues. The cytoplasmic [3H]Oe binding in the studied tissues seems to be affected by the naloxone binding system. After in vitro saturation of naloxone binding sites by naloxone the [3H]Oe binding to low affinity sites (type II) in hypothalamus as well as in uterus has been increased by 8- and 2-fold, respectively. These results indicate the presence of specific [3H]Nal binding in rat uterus with similar properties to those found in the hypothalamus. Furthermore an interaction between opioid and oestradiol receptor systems could be also suggested.     

Effects of ovariectomy and estradiol replacement on naloxone induced LH secretion in the female rabbit

The effects of an opioid antagonist, naloxone, on the secretion of gonadotrophins were investigated in the long term ovariectomized rabbit. In the intact and acutely ovariectomized rabbit (2 days p.o.) naloxone at 10 mg/kg induced an increase of 260-300% in LH secretion at 40 min post-injection. From days 33-66 post-surgery naloxone at 10 mg/kg caused significant elevations in LH release even when animals were treated with estradiol benzoate 24 h previously. By contrast, treatment with oestradiol benzoate 3 h before naloxone abolished the LH increase. An LH surge could be elicited in these rabbits with GnRH treatment. These studies indicated that long term ovariectomy in the female rabbit does not completely remove the opioid control of GnRH release and that the LH response to naloxone is influenced by circulating estradiol levels.     

Effect of dexamethasone on uterine cell death

Oestrogen, progesterone and androgen inhibit uterine cell death after the depletion of oestrogen. In the present study, we investigated effects of glucocorticoid on death of mouse uterine cells. Castrated female mice were given a daily injection of 17 beta-oestradiol (0.2 microgram/mouse/day) for 3 days, and then an injection of 5'-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) to label DNAs of uterine cells with 125I. Mice were killed at intervals during subsequent treatments, and the retention of [125I]IdUrd incorporated into the whole uterus was determined. On subsequent injection of vehicle only, the 125I-radioactivity retained in the whole uterus rapidly decreased. Injections of dexamethasone (50 micrograms/mouse/day) reduced the loss of 125I-radioactivity slightly but significantly. Dexamethasone also showed synergistic effects on the retention of 125I-radioactivity when it was daily injected together with 17 beta-oestradiol, progesterone or 5 alpha-dihydrotestosterone. The present results suggest that glucocorticoid may affect the processes involved in the uterine cell death, in a manner such as inhibiting the uterine cell death or delaying the removal of DNAs of dead cells from the uterus.     

Synthesis of a potent enkephalin analog, Tyr-D-Thr-Gly-Phe-delta 3 Pro-NH2, and some of its biological activities

The enkephalin analog, H-Tyr-D-Thr-Gly-Phe-delta 3 Pro-NH2 X HCl (VI) ([D-Thr2, delta 3 Pro5]-enkephalinamide), has been synthesized by conventional methods in solution and purified to homogeneity by reversed phase HPLC. The analog is a potent analgesic agent. For evaluation of some of its biological activity a related compound [D-Thr2, Thz5]-enkephalinamide (XI) was also synthesized in solution. Anti-diarrhea activity was evaluated in mice by the intravenous route for anti-DL-5-hydroxytryptophan (5-HTP) induced diarrhea activity. Analgesic activity was assayed by the method of Nilsen in mice using the intravenous route, and by a modified tail flick test in rats and the acetic acid writhing test in mice following subcutaneous administration. Within the constraints of the assays the two analogs are approximately equipotent. Both are less active than [D-Ala2, MePhe4, Met(0)ol]-enkephalinol (XII). Earlier receptor binding studies of compound XI indicated enhanced affinity for the mu receptor and little for the delta receptor. By comparison this may also be the case for compound VI.     

Effects of actinomycin D and estrogen priming on decidual growth in ovariectomized mice.

Ovariectomized mice were primed for 2 days with estradiol and/or actinomycin D. In order to evaluate the effects of these treatments on endometrial cell proliferation, colchicine and [3H]-thymidine were administered shortly before killing groups of animals at days 4 and 5 after priming (the latter groups received 500 micrograms progesterone plus 10 ng estradiol 24 h before killing). The same priming treatments were administered 3 days before starting hormonal treatment eliciting uterine sensitivity to decidualization (incuded by intrauterine oil injection). The comparison of labeling and mitotic indices and of decidual tissue weights between experimental groups showed that under such conditions: (1) actinomycin D only partly inhibits the effects of estrogen priming: the increase in cell division obtained on day 4 after priming remains unchanged in all three endometrial components while the increase in stromal mitotic activity at day 5 and further decidual growth are reduced, (2) since the inhibition rate of these parameters is greater in non-primed than in primed animals, it appears that estrogen priming counteracts the antagonistic action of actinomycin D.