The effects of cholinergic compounds on the development of morphine tolerance, dependence and increased naloxone potency in mice

The effects of chronic treatment with morphine and cholinergic compounds on the development of morphine tolerance, physical dependence and increased naloxone potency were studied. Using the abdominal constriction method, it was shown that morphine tolerance was apparent after s.c. administration of morphine 20.0 mg/kg three times a day for four days. It was found that, in animals which showed a low degree of morphine tolerance, the naloxone potency was similar to that determined in mice which had been pretreated with only a single dose of morphine which causes no measurable tolerance. Thus, the development of increased naloxone potency and tolerance to morphine do not parallel each other. In addition, while atropine inhibited, and anti-cholinesterase drugs enhanced, the development of increased naloxone potency caused by morphine treatment they had no or little effect on the development of morphine tolerance. Furthermore, chronic treatment with cholinergic agonists reduced, while muscarinic antagonist enhanced, the development of physical dependence on morphine as assessed by withdrawal jumping and body weight loss. It is concluded that the increased potency of naloxone in antagonising the antinociceptive effect of morphine can be dissociated from the development of tolerance to, and physical dependence on, morphine in mice.     

Inhibition of morphine-induced tolerance and dependence by a benzodiazepine receptor agonist midazolam in the rat

We investigated whether midazolam administration influenced morphine-induced antinociception and tolerance and dependence in the rat. Antinociception was assessed by the tail-flick (TF) and the hot-plate test (HP 52 degrees C). Morphine tolerance developed after daily single injections of morphine for 11 days. The effect of midazolam on morphine-induced antinociception and tolerance was assessed by giving daily injections of various doses of midazolam for 11 days. The first injection of saline or midazolam was given intraperitoneally and 30 min later morphine (10 mg/kg body weight) was administered subcutaneously. Antinociception was monitored by measuring TF and HP latencies 60 min after the second injection. Midazolam was injected at four different concentrations: 0.03, 0.1, 0.3, and 3 mg/kg body weight. Chronic administration of morphine resulted in the development of tolerance to antinociception in both TF and HP tests, with rats exhibiting baseline antinociception on Day 9. Animals treated with midazolam alone showed little antinociception on Days 3-9. However, midazolam administration in morphine-treated animals attenuated morphine-induced tolerance to antinociception on Days 1-11 as measured by the tail-flick test. Midazolam also decreased the jumping behavior following naloxone injections in morphine-dependent rats. These results suggest that midazolam may prolong the effects of morphine by delaying morphine-induced development of tolerance to antinociception. Midazolam also attenuated a decrease in weight gain induced by chronic injections of morphine.     

An intraventricular infusion model for inducing morphine dependence in rats: Quantitative assessment of precipitated withdrawal.

Describes an experimental model of morphine dependence in which male Sprague-Dawley rats were made dependent upon morphine by intraventricular infusion. Morphine dependence was quantified by a series of withdrawal signs that were induced by the intraperitoneal administration of the morphine antagonist naloxone (5 mg/kg). The infusion of different concentrations of morphine resulted in the production of physical dependence, the severity of which was directly correlated with the concentration of morphine infused. A weak to moderate degree of dependence characterized by such withdrawal signs as teeth chattering, whole-body shakes, and vocalization was produced by infusions of morphine less than 5 μg/hr. A strong degree of physical dependence characterized by additional dominant withdrawal signs such as jumping and launching was produced by the infusion of 50 μg/hr morphine. The morphine pellet model that most closely approximated this degree of dependence was a 3-pellet model in which a single 75-mg morphine pellet was implanted at 48-hr intervals. Abstinence precipitated by removal of the morphine-containing osmotic minipumps was characterized primarily by teeth chattering and whole-body shakes that persisted for at least 48 hrs. (38 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Characterization of the membrane-associating domain of the sperm adhesive protein, bindin

Male Swiss-Webster mice were rendered tolerant to morphine by subcutaneous implantation of a morphine pellet, each containing 75 mg morphine base, for 3 days. Mice implanted with placebo pellets served as controls. A high degree of tolerance to the analgesic effect of morphine developed as evidenced by decreased analgesic response to various doses of morphine. A selective kappa-opiate agonist, U-50,488H (8, 16 and 32 mg/kg, i.p.) produced dose-dependent analgesic and hypothermic effects in mice implanted with placebo pellets. A significant decrease in the analgesic and hypothermic effects of U-50,488H was observed in morphine tolerant mice as compared to placebo-treated mice. Mice were rendered tolerant to U-50,488H by injecting the drug (25 mg/kg, i.p.) twice daily for 4 days. Vehicle injected mice served as controls. Tolerance to the analgesic and hypothermic effects of U-50,488H in mice injected chronically with the drug was evidenced by the decreases in the intensity of these responses when compared to those observed in vehicle injected controls. Morphine produced a dose-dependent analgesic and hypothermic effects in mice injected chronically with vehicle but the intensity of these effects was significantly lower in mice injected chronically with U-50,488H. These results indicate that a substantial tolerance to analgesic and hypothermic effects of U-50,488H develops in morphine tolerant mice. The effect of chronic injections of U-50,488H on the binding of [3H]ethylketocyclazocine (EKC) and [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (DAMGO) to whole brain and spinal cord kappa- and mu-opiate receptors was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial loss of tolerance liability to morphine analgesia in mice lacking the nociceptin receptor gene

In mice lacking the nociceptin (or orphanin FQ) receptor gene, when 10 mg/kg of morphine was subcutaneously given, a potent analgesia in the tail pinch test was observed. The analgesic effect of morphine was equivalent among wild-type, heterozygous and homozygous mutant mice. When morphine was given to such mice in a dose of 10 mg/kg once per day for 5 days, wild-type and heterozygous mice showed marked tolerance or reduction in the morphine analgesia on the 5th day, while homozygous mice showed only 50% reduction in the peripheral analgesia of morphine. These findings suggest that nociceptin or its receptor plays important roles in the in vivo mechanism for the development of morphine tolerance.     

Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles

We have recently reported that the short-acting anesthetic and analgesic drug midazolam can produce analgesia and decrease morphine tolerance and dependence in the rat by interacting with the opioid system. This study was designed to investigate the effect of midazolam, morphine, and both together on met-enkephalin levels in the rat. Male Sprague-Dawley rats were divided into four groups: (1) saline-saline; (2) saline-morphine; (3) midazolam-saline, and (4) midazolam-morphine groups. First, a saline or midazolam injection was given intraperitoneally and after 30 min a second injection of saline or morphine was given subcutaneously once daily for 11 days. Animals were sacrificed on the 11th day 60 min after the last injection to measure met-enkephalin by radioimmunoassay. Morphine tolerant animals showed a significant increase in met-enkephalin levels in the cortex (137%) and midbrain (89%), and a significant decrease in met-enkephalin levels in the pituitary (74%), cerebellum (34%) and medulla (72%). Midazolam treated animals showed a significant decrease in met-enkephalin levels in the pituitary (63%), cortex (39%), medulla (58%), kidneys (36%), heart (36%) and adrenals (43%), and a significant increase in met-enkephalin levels in the striatum (54%) and pons (51%). When morphine and midazolam were injected together, midazolam antagonized the increase in met-enkephalin levels in cortex and midbrain region and the decrease in met-enkephalin level in the medulla region observed in morphine tolerant animals. These results indicate that morphine tolerance and dependence is associated with changes in the concentration of met-enkephalin in the brain. Midazolam may inhibit morphine tolerance and dependence by reversing some of the changes induced in met-enkephalin levels in brain by morphine in morphine tolerant and dependent animals.     

Effect of pargyline on morphine tolerance and physical dependence development in mice

The effects of single and repeated pargyline administration on morphine antinociception in both naive and morphine-tolerant mice and on naloxone-precipitated withdrawal in morphine tolerant-dependent animals were investigated. Adult, male Swiss-Webster mice were rendered tolerant to and dependent on morphine by the s.c. pellet implantion technique. Morphine analgesia, as assessed by the tail-flick antinociceptive test, was potentiated in tolerant animals by acute adminstration of pargyline but antagonized by repeated pargyline administration; pargyline produced similar effects in non-tolerant mice and to the same relative degree. Repeated pargyline treatment during morphine pellet implantation enhanced the withdrawal jumping response precipitated by naloxone in dependent mice. Pargyline also, after a single injection, exacerbated jumping in mice undergoing abrupt withdrawal. Neither acute nor chronic pargyline administration altered the brain distribution of injected morphine in non-tolerant mice. It was concluded that pargyline may modify acute morphine actions and withdrawal without materially altering the process(es) involved in the development of tolerance and physical dependence.     

Opioid receptors altered binding nature in guinea-pig brain following the development of morphine dependence

Morphine is well known to produce tolerance and dependence. The mechanisms for these phenomena are not clearly understood and there are a number of conflicting reports that chronic morphine administration decreases, increases, or leaves unchanged the number of opioid binding sites. We examined the potency of MScontin (oral controlled-release preparation of morphine) to induce morphine dependence and also determined the change of mu, delta and kappa opioid receptor types in brain homogenates obtained from morphine-dependent guinea-pigs. 1. Guinea-pigs were implanted subcutaneously with MScontin (300 mg.kg-1) and naloxone was employed to precipitate jumping behavior of withdrawal symptoms at various times. The highest degree of physical dependence was observed on the 2nd day after implantation. Therefore, this period was chosen to investigate opioid receptor binding assay. 2. Two days after implantation, the binding of 3H-DAGO (mu agonist), 3H-DPDPE (delta agonist) and 3H-U69593 (kappa agonist) to brain membranes prepared from morphine dependent and control guinea-pigs was determined. Scatchard plot of the saturation binding data revealed an increase in Bmax values (maximum specific binding) and no change in the Kd values (equilibrium dissociation constants) of 3H-opioid ligand bindings obtained from morphine-dependent animals as compared to controls. These results indicate that brain mu, delta and kappa opioid receptors are up-regulated in morphine dependent guinea-pigs.     

Naloxone-induced opiate withdrawal produces long-lasting and context-independent changes in aggressive and social behaviors of postdependent male mice.

The purpose of this study was to determine whether an environment associated with naloxone-induced morphine withdrawal affects aggressive or social behaviors in postdependent mice. Morphine-dependent or saline-treated mice received 3 naloxone injections in 1 of 2 different environments (A or B); 15 days afterward, when the mice were completely drug free, an aggression test was carried out in Environment A. All the mice suffering morphine withdrawal showed a significant increase in aggression, irrespective of the environment in which the withdrawal took place. In these conditions, the impact of morphine dependence and the 3 induced withdrawals was so profound that the environment could not be discriminative. In addition, modifications in the behavioral profile of postdependent mice that suffered only spontaneous withdrawal were long-lasting, with the mice carrying out more attacks during social investigation without presenting threat postures. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Ontogeny of morphine withdrawal in the rat.

The present studies examined behavioral changes during precipitated morphine withdrawal in 7- to 42-day-old rat pups. One group of rats was injected with morphine sulfate (10.0 mg/kg) twice daily for 6.5 days. Another group of 7-day-old rats received a lower dose of morphine (3.0 mg/kg). Controls were saline injected or untreated litters (7-day-old pups only). On Day 7, a target pup was injected with saline or naltrexone (0.3–20.0 mg/kg). Preweaning pups were observed in a warm chamber with the litter. Forty-two-day-old rats were tested individually. Morphine-treated pups tested with naltrexone showed significant alterations in behavior that varied at different ages. For example, rolling, stretching, and head and paw moves were observed at the younger ages, whereas burrowing, diarrhea, jumps, teeth chatter, and wet dog shakes occurred in the older rats. These data indicate that morphine-abstinent rats demonstrate withdrawal signs that are within the developmental repertoire of the rat. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Physical dependence produced by dihydroetorphine in mice

Using various administration schedules, the physical dependence produced by dihydroetorphine (DHE) was compared with that of morphine in mice. Physical dependence, evaluated by naloxone-precipitated withdrawal signs, did not develop following daily treatment with DHE (10, 20, 100 and 1000 micrograms/kg, i.p. or 30, 100 and 1000 ng/mouse, i.c.v.) for 6 d. However, 5 repeated injections of DHE (10 micrograms/kg, i.p.) at 1 or 2 h intervals did produce physical dependence and the dependent state disappeared after 2 h. Accordingly, it was demonstrated that a sufficient degree of antinociceptive activity needed to be maintained, longer than several hours, for the development of physical dependence on DHE and that the duration of the dependent state was very short. In the single dose suppression test, a single dose of DHE completely suppressed the natural withdrawal signs that appeared following abstinence in morphine-dependent animals without reappearance of significant withdrawal signs, indicating the suitability of DHE as a substitute for morphine. The characteristic properties of DHE, the extremely potent antinociceptive effect and minimal dependence, indicate the separation of the antinociceptive effect from dependence, and suggest that it may be possible to develop a novel drug which may be safely used in clinical situations.     

Biochemistry and genetics of thiodicarb resistance in the house fly (Diptera: Muscidae)

We have recently reported that environmental toxicants, such as DDT, PCBs, pyrethroids, and nicotine can induce permanent functional and neurochemical changes in adult mice when given to neonatal mice during the peak of rapid brain growth. In the present investigation the neurotoxic effects following neonatal exposure to paraquat (N,N'-dimethyl-4,4'-bipyridylium), a broad-spectrum herbicide with structural similarity to the 1-methyl-4-phenylpyridium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which can induce Parkinson's syndrome, and MPTP were studied. Five groups of mice were given paraquat or MPTP orally: group 1, vehicle; groups 2 and 3, MPTP 0.3 and 20 mg/kg; groups 4 and 5, paraquat 0.07 and 0.36 mg/kg when 10 and 11 days old. Neonatal spontaneous motor activity was tested on Day 18 in mice given paraquat 0.36 mg/kg body wt. Adult spontaneous motor activity testing was performed at ages 60 and 120 days. On Day 125 the mice were decapitated and the contents of dopamine (DA), serotonin (5-HT), and metabolites in striatum were analyzed. The results may be summarized as follows: (1) No signs of acute toxicity or differences in weight gain were observed in any of the groups. Nor was any respiratory distress or motor performance dysfunction evident on Day 18 in mice given paraquat 0.36 mg/kg body wt. (2) The behavioral tests at 60 days of age showed a marked hypoactive condition in the mice given paraquat (at both doses) and MPTP (at both doses). (3) At the age of 120 days the hypoactive behavior persisted and appeared even more pronounced. (4) The high doses of MPTP and paraquat--and to a less extent the low doses--reduced the striatal content of DA and metabolites without affecting 5-HT. The altered behavior, together with the dose-dependent reduction of DA and metabolites in neostriata in this study, further demonstrates the susceptibility to low-dose exposure to environmental pollutants during the neonatal period.     

Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine

Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKC gamma) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of the N-methyl-D-aspartate receptor antagonist MK-801 on tolerance-associated PKC gamma changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 micrograms morphine for eight days, PKC gamma immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKC gamma immunostained neurons in laminae I-II than in laminae III-IV and V-VI. Such PKC gamma immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKC gamma increases. Moreover, both the development of morphine tolerance and the increase in PKC gamma immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKC gamma immunoreactivity was not increased in rats receiving a single i.t. administration of 10 micrograms morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKC gamma immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.     

Performance and tissue zinc and metallothionein accumulation in chicks fed a high dietary level of zinc

Four experiments were conducted to identify several factors that might improve the accuracy and reproducibility of Zn bioavailability assays for chicks. Response of tissue Zn and metallothionein (MT) concentrations to various elevated levels and soluble sources of dietary Zn were measured, as well as the effect of delaying high Zn administration until 7 d posthatching to alleviate the detrimental effect of Zn sulfate on feed intake to 3 wk of age. Bone Zn increased (P < 0.01) in all experiments in response to increasing dietary Zn concentrations. Liver and pancreas MT were affected (P < 0.01) by a source by age interaction and variability that made this criterion unsuitable for bioavailability assays. Lastly, 1-d-old chicks were used to study the effect of delaying feeding of a high-Zn diet up to 7 d of age. The basal diet was fed continuously for 21 d as a control. A diet containing 1,000 ppm Zn was either fed continuously from Day 1, or started on Day 3, 5, or 7. Chicks given high Zn on Day 3, 5, or 7 decreased (P < 0.01) feed intake within 24 h of feeding. Delayed feeding of high dietary Zn might help to alleviate decreased feed intake observed in previous studies. Delaying the onset of high Zn feeding by several days may help alleviate feed intake problems observed with Zn sulfate. Use of either Zn gluconate or Zn acetate as a standard in assays or use of MT synthesis as a bioavailability criterion will probably not be useful to improve accuracy of the estimates.     

Morphine analgesia: Enhancement by shock-associated cues.

Recent research has shown that rats exposed to repeated stress display enhanced morphine analgesia. Six experiments, with 218 male Sprague-Dawley rats, examined the possible contribution of classically conditioned analgesia to this effect. Drug-naive Ss exposed to 9 daily sessions of stress, each consisting of a single exposure to footshock, subsequently displayed enhanced analgesic responsiveness to morphine sulfate (5 mg/kg, sc) 1 and 10 days after stress. This enhancement was also observed in morphine-experienced Ss 1 and 8 days after stress. The effect of footshock stress on morphine analgesia was specific to the environment in which stress was administered. Conditioned analgesia was found under the same conditions that yielded enhanced morphine analgesia, and both this conditioned analgesia and the acute analgesia elicited by the footshock stressor were attenuated by naloxone (20 mg/kg). Data are consistent with the hypothesis that the enhanced morphine analgesia observed after repeated footshock stress reflects the contribution of an opioid-mediated, conditioned analgesia elicited by cues formerly paired with the stressor. (54 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modelling migration: the clock-and-compass model can explain the distribution of ringing recoveries

The present study was carried out to investigate the relative involvement of spinal opioid receptors in the development of physical dependence on intrathecal (i.t.) butorphanol in comparison with i.t. morphine. Dependence was induced by continuous i.t. infusion of butorphanol (52 nmol/h) and morphine (26 nmol/h) for 4 days in male Sprague-Dawley rats. Naloxone, CTOP, naltrindole, and nor-binaltorphimine (nor-BNI) were administered i.t. to precipitate behavioral signs of withdrawal. Administration of i.t. naloxone produced a significantly greater increase in the profile of withdrawal signs in i.t. morphine dependence than that in i.t. butorphanol dependence. An i.t. nor-BNI challenge elicits behavioral signs of withdrawal only in rats dependent on i.t. butorphanol, but not in rats dependent on i.t. morphine. CTOP administered i.t. precipitated withdrawal signs in i.t. morphine dependence that were greater than that in i.t. butorphanol dependence. An i.t. treatment with naltrindole produced equivalent signs of withdrawal in both i.t. butorphanol- and morphine-dependent rats. These results suggest that continuous i.t. butorphanol results in the development of less physical dependence than that of i.t. morphine. Spinal kappa- rather than delta- and mu-opioid receptors play a major role in the development of i.t. butorphanol dependence, whereas spinal mu-opioid receptors play a more important role than delta-opioid receptors in i.t. morphine dependence.     

Effects of caerulein and CCK antagonists on tolerance induced to morphine antinociception in mice

Different groups of mice received one daily dose (50 mg/kg) of morphine subcutaneously (SC) for 3, 4 or 5 days to develop tolerance to the opioid. The antinociceptive response of morphine (9 mg/kg) was tested in the hot-plate test 24 h after the last dose of the drug. Tolerance to morphine was obtained in all groups. The group of mice that received morphine for 4 days was employed for the rest of the experiments. Pretreatment of animals with a single dose of caerulein (0.025, 0.05, and 0.1 mg/kg, SC) 30 min prior to receiving morphine (50 mg/kg; during the development of tolerance to the opioid) on day 1, 2, 3, 4 or 5 of morphine administration potentiate antinociception induced by morphine (test dose of 9 mg/kg). The dose of 0.05 mg/kg of caerulein, used 30 min before morphine administration on day 3, was also used to evaluate the effects of antagonists on caerulein-induced decrease in tolerance. The selective cholecystokinin (CCK) receptor antagonists, MK-329 [1-methyl-3-(2 indoloyl)amino-5-phenyl-3H-1,4-benzodiazepin-2-one; 0.25 and 0.5 mg/kg] or L-365,260 [3R(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H- 1,4-benzodiazepin-3-yl)-N-(3-methyl-phenyl)urea: 0.25 and 0.5 mg/kg] decreased potentiation of morphine response induced by caerulein. MK-329 or L-365,260, when were injected 35 min before morphine injection during the development of tolerance and on day 3, decreased the tolerance to morphine. A single administration of MK-329 or L-365,260 (in the absence of caerulein) 35 min and 48 h before the test dose of morphine (9 mg/kg) potentiated the antinociception of morphine in nontolerant animals. In conclusion, CCK mechanism(s) may interact with morphine tolerance.     

Identification of human and mouse GP-1, a putative member of a novel G-protein family

OBJECTIVES: To determine a) if serum morphine concentration changes during the first 3 hrs of extracorporeal membrane oxygenation (ECMO); and b) if absorption of morphine onto the membrane oxygenator is responsible for these changes. Also, morphine clearance during the first 5 days of ECMO was studied. DESIGN: Prospective, open-label study with consecutive patient enrollment. SETTING: Neonatal intensive care unit at a university-affiliated, children's hospital. SUBJECTS: Eleven neonates with severe persistent pulmonary hypertension of the newborn receiving continuous intravenous infusions of morphine sulfate and requiring ECMO. INTERVENTIONS: Blood samples were obtained from the subjects and ECMO circuits at predetermined time intervals. MEASUREMENTS AND MAIN RESULTS: Serum morphine concentration was determined using high-performance liquid chromatography. Morphine concentrations were no different from baseline at 5 mins, 1 hr, or 3 hrs after beginning ECMO. There was no significant difference in morphine concentration from samples taken immediately proximal and distal to the membrane oxygenator at 5 mins, 1 hr, and 3 hrs after the start of ECMO. Morphine clearance was calculated on days 1, 3, and 5 of ECMO. The mean value for morphine clearance was 11.7 +/- 9.3 (SD) ml/min/kg (range 2.6 to 34.5). CONCLUSIONS: The initiation of ECMO does not lead to a significant decrease in serum morphine concentration and there is no uptake of morphine onto the membrane oxygenator of the ECMO circuit. Morphine clearance for infants receiving ECMO is variable.     

Naloxone-induced supersensitivity of oxytocin neurones to opioid antagonists

Here we report that a single administration of naloxone to conscious rats produces no significant increase in oxytocin release, but when repeated 3-4 days later results in a large release of oxytocin. Plasma oxytocin concentrations were measured in conscious and urethane-anaesthetized rats pretreated with naloxone or isotonic saline on Day 1. On Days 2, 3 or 4, a second dose of naloxone was given, producing an increase in oxytocin secretion in naloxone-pretreated groups (P < 0.05 vs. controls) on Day 3 and 4, but not on Day 2. The specificity of the opioid antagonist supersensitivity was determined by injection of the kappa-antagonist nor-binaltorphimine (nor-BNI). Pretreated rats (naloxone, saline or nor-BNI, Day 1) received an additional acute nor-BNI injection (Day 4) which increased plasma oxytocin concentration in the three groups. However, this increase was higher in naloxone-pretreated rats with no differences between the nor-BNI- and saline-pretreated animals. Measurements of electrical activity of single supraoptic nucleus oxytocin neurons and of plasma oxytocin concentration (Day 4) showed that naloxone modestly enhanced the responsiveness of oxytocin neurons to cholecystokinin (CCK) in naloxone-pretreated rats (by comparison with saline-pretreated rats), but had only a small effect on basal firing rate that did not differ between naloxone-pretreated rats and saline-pretreated rats. To investigate whether naloxone-pretreatment modified the effect of morphine on CCK-induced oxytocin release, on Day 4 CCK was injected i.v. with or without morphine. Morphine at a dose of 0.1 mg/kg did not affect CCK-induced oxytocin release, whereas 1 mg/kg of morphine blocked this release in both saline- and naloxone-pretreated rats. The results suggest that naloxone induces opioid antagonist supersensitivity on oxytocin secretion, mainly by up-regulating kappa-opioid mechanisms on oxytocin nerve terminals in the posterior pituitary.     

Biosynthesis of glycosaminolgycans during corneal development

Glycosaminoglycan biosynthesis was studied in developing chick corneas, with particular attention paid to keratan sulfate I, the major glycosaminoglycan of this tissue. This polysaccharide is unique to the cornea and may be required for the development and maintenance of corneal transparency. Corneas from 5-to 20-day chick embryos were labeled in vitro with D-[6- 3H] glyhucosamine and H(2)35SO(4)35SO(4) and the amount of label in each glycosaminoglycan was determined. The data indicate that, contrary to previous suggestions, keratan sulfate biosynthesis in the cornea begins at the time of fibroblast invasion of the primary stroma, at least 8 days prior to the onset of corneal transparency, which occurs on Day 14 of the development in the chick. The rate of incorporation of radioactivity into keratan sulfates, on a dry weight basis, increases rapidly after Day 6 and levels off on Day l4. The proportion of 3H and 35S in keratan sulfate reaches nearly maximal levels as early as Day 9. In contrast, the proportion of radioactivity in corneal heparan sulfates declines rapidly after Day 5. However, the rate of incorporation of radioactivity into heparan sulfates, on a dry weight basis, increases or remains the same during early development. On and after Day 14, keratan sulfates appear to become more highly sulfated. Moreover, the ratios of 4-sulfated to 6-sulfated chondroitin sulfates increase during development, reaching a maximum on Day 14. These changing patterns of glycosaminoglycan biosynthesis during corneal development may play an important role in corneal morphogenesis and the achievement of corneal transparency