Influence of clonidine on the acute dependence response elicited in naive rats by naloxone

Clonidine has been used successfully in the treatment of opiate dependence. The discomforting effects of withdrawal are attenuated by the drug. The question of whether the more central process of dependence is affected by clonidine was tested in the present study. Change in plasma corticosterone was used as the indication of the stress of acute withdrawal from morphine. Conscious, unrestrained male rats showed a dose-related, though somewhat delayed, increase in plasma corticosterone after clonidine (0.01-0.1 mg/kg). The suggested mechanism for this effect involves presynaptic inhibition of noradrenergic neurons inhibiting CRF (corticotropin-releasing factor) release. Similar animals showed an elevation of plasma corticosterone after naloxone (0.4 mg/kg) was administered 3 hrs following a single morphine-priming (10 mg/kg). The naloxone-precipitated response was unaffected by clonidine (0.04 mg/kg). This dose of clonidine did not substitute for morphine-priming to produce the naloxone-precipitated response. The data suggests that clonidine elevated plasma corticosterone by an indirect mechanism. Further, the stress associated with acute withdrawal is unaffected by clonidine suggesting that the drug does not alter dependence development.     

Lack of correlation between naloxone-induced changes in sexual behavior and serum LH in male rats

Four experiments were conducted to determine whether the action of opiate receptor antagonist drugs on sexual performance in male rats is mediated by the central release of luteinizing hormone releasing hormone (LHRH). First, in Experiment 1 it was demonstrated that administration of naloxone (20 mg/kg) caused a lengthening of postejaculatory intervals and an elevation of serum LH concentrations in gonadally intact male rats. In Experiment 2, manipulation of females' proceptive and receptive behaviors failed to reveal the reductions in ejaculation latencies and in the number of intromissions preceding ejaculation which have been previously reported after administration of naloxone to male rats. Again, the predominant response to treatment with naloxone was an increase in the length of the postejaculatory interval. In Experiment 3, pinching the tails of male rats every 30 sec after ejaculation partially abolished the relative refractory periods of the postejaculatory intervals; naloxone-induced increases in the lengths of these shortened postejaculatory intervals were nevertheless identical to those of control males, suggesting that naloxone acts to lengthen the absolute refractory period. Finally, in Experiment 4 naloxone was given to castrated males implanted with testosterone-filled silastic capsules ranging in length from 2 to 45 mm, which produced a wide range of basal serum LH concentrations. Naloxone caused an increase in postejaculatory intervals; however, this effect was not correlated with the degree to which naloxone stimulated serum LH, suggesting that the effects of naloxone on the postejaculatory interval are not mediated by a drug-induced release of LHRH.     

Naloxone doesn't determine the response of growth hormone to clonidine in obese patients

The effect of naloxone (opioid receptor blocker) on the impairment of growth hormone (GH) release after clonidine (alfa 2-adrenergic agonist) was investigated in 10 volunteer obese subjects. The patients (4 males and 6 females, 16-22 year old) with fat excess (15 +/- 2 kg) estimated by bioelectrical impedance analysis (BIA) were studied repeatedly. The patients, were perfused by a slow saline infusion. 30 min later they received a bolus dose of clonidine (150 micrograms p.o.), followed 30 min later by a bolus dose of naloxone (10 mg i.v.) or a corresponding volume of isotonic sodium cloride (I.S.) for control. No significant changes occurred in blood GH concentration after clonidine administration and naloxone did not induce GH response at clonidine. These results suggest that in obese subjects the impairment of GH release after clonidine is not mediated via receptors sensitivity to naloxone.     

Effects of (D-ala2, met5)-enkephalinamide and naloxone on ACTH and corticosterone secretion

An intravenous administration of (D-ala2, met5)-enkephalinamide (DALA) caused a significant elevation of plasma ACTH and corticosterone at 10 to 20 min after injection in unanesthetized freely moving rats. An intraperitoneal administration of cyproheptadine tended to reduce plasma ACTH and corticosterone levels at 60 min after injection, but it did not attenuate the DALA-induced ACTH and corticosterone elevation. A large dose of naloxone (1-10 mg/kg body weight) caused a significant elevation in plasma corticosterone, but naloxone at 10 mg/kg body weight reduced the basal ACTH level and DALA-induced ACTH elevation. When both DALA and naloxone were injected, the steroidogenic effect was attenuated. Neither DALA nor naloxone affected the basal ACTH release and CRF-induced ACTH stimulation in rat anterior pituitary cell cultures. These results suggest that DALA acts at the extra-hypophyseal level to stimulate ACTH and corticosterone and that the naloxone stimulatory effect on steroidogenesis acts on the adrenal gland or is mediated by stimulating corticosterone stimulating factors other than ACTH.     

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.     

Further evidence that naloxone acts as an inverse opiate agonist: Implications for drug dependence and withdrawaL

To test if naloxone behaved as an inverse agonist rather than as an antagonist we evaluated its responses in guinea-pig ilea with and without morphine (480 nM, 24 h). In control ilea, naloxone (100 nM) had no effect. In morphine-treated ilea, naloxone as a bolus, but not as an infusion, elicited an abstinence response. Preadministration of naloxone blocked the response to subsequent administrations. Similarly, naloxone failed to produce an abstinence response in ilea pretreated with kappa compounds (bremazocine, U50488 or xorphanol 100 nM) or with kinase inhibitors (H7 or H8 30 μM). These findings can be interpreted in the light of the two-state receptor model if naloxone behaves as an inverse agonist: Incubation with morphine increased the active state of receptors making them susceptible to the inverse agonist (naloxone); exposure to naloxone favored the inactive conformation making them insensitive to further administration of naloxone; kappa compounds behaved as antagonists preventing the response to naloxone; and kinase inhibitors interfered with the active conformation making the system insensitive to naloxone. According to this model, dependence can be viewed as an overexpression of the active receptors and withdrawal as an abrupt change from the active to the inactive state.     

Induction of riboflavin-carrier protein in the immature male rat by estrogen: Kinetic and hormonal specificity

The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.     

Peripheral motilin administration stimulates feeding in fasted rats

Although the physiologic function of the gastrointestinal hormone motilin remains uncertain, plasma levels of this peptide vary with migrating myoelectric complexes (MMCs) in the small intestine. In the fed state, both MMCs and plasma motilin are suppressed. During fasting, cyclical peaks of motilin in plasma occur at the same time as Phase III of the MMC cycle occurs in the duodenum. This dependence of motilin concentrations in plasma on the feeding state of the animal prompted an investigation of the effects of motilin on feeding behavior. Intraperitoneal injection of motilin into fasted, but not fed, rats stimulated eating in a dose dependent manner. A significant stimulation of feeding was seen at doses of 5 and 10 μg/kg. Sated rats did not eat whether injected with motilin or vehicle. The feeding response to motilin was blocked by prior injection of the rats with naloxone, naltrexone, or pentagastrin. The dose response suppression of food intake by naloxone was similar in fasted animals treated with motilin or vehicle. Motilin may function as a hunger hormone during periods of fasting.     

Opioidergic control of luteinizing hormone secretion in the female rabbit: influence of age on the response to naloxone

To examine the role of opioid neurons on luteinizing hormone (LH) secretion in the female rabbit, we determined LH release at timed intervals after naloxone administration to rabbits aged 25-150 days. The LH response to naloxone (10 mg/kg) was not significantly elevated until day 43 when LH rose 76-113% above basal levels at 40-80 min. In 56-day-old females the corresponding increase was 160% at 15 min and in 65- to 67-day-olds it was 154%. From 70 to 80 days of age the LH response was blunted and no significant elevations could be elicited. By contrast, naloxone-induced LH increases were again evident when rabbits were older than 100 days. At all ages no significant change in FSH concentrations was observed. In the adult females, naloxone at 2.5, 5, and 10 mg/kg caused increases in LH secretion which occasionally were high enough to induce ovulation as exemplified by elevated serum progesterone 4 days later. These data suggest that opioid peptides may be involved in the prepubertal rise in LH and in the normal inhibition of adult secretion in the female rabbit.     

Opioid regulation of luteinizing hormone secretion in the male rat

Current evidence suggests that endogenous opioid peptides (EOPs) tonically inhibit secretion of luteinizing hormone (LH) by modulating the release of gonadotropin-releasing hormone (GnRH). Because of their apparent inhibitory actions, EOPs have been assumed to alter both pulse frequency and amplitude of LH in the rat; and it has been hypothesized that EOP pathways mediate the negative feedback actions of steroids on secretion of GnRH. In order to better delineate the role of EOPs in regulating secretion of LH in the male rat, we assessed the effects of a sustained blockade of opiate receptors by naloxone on pulsatile LH release in four groups: intact male rats, acutely castrated male rats implanted for 20 h with a 30-mm capsule made from Silastic and filled with testosterone, acutely castrated male rats implanted for 20 h with an osmotic minipump dispensing 10 mg morphine/24 h, and male rats castrated approximately 20 h before treatment with naloxone. We hypothesized that if EOPs tonically inhibited pulsatile LH secretion, a sustained blockade of opiate receptors should result in a sustained increase in LH release. We found that treatment with naloxone resulted in an immediate but transient increase in LH levels in intact males compared to controls treated with saline. Even though mean levels of LH increased from 0.15 +/- 0.04 to a high of 0.57 +/- 0.14 ng/ml, no significant difference was observed between the groups in either frequency or amplitude of LH pulses across the 4-h treatment period. The transient increase in LH did result in a 3- to 4-fold elevation in levels of plasma testosterone over baseline. This increase in testosterone appeared to correspond with the waning of the LH response to naloxone. The LH response to naloxone was eliminated in acutely castrated rats implanted with testosterone. Likewise, acutely castrated rats treated with morphine also failed to respond to naloxone with an increase in LH. These observations suggest that chronic morphine and chronic testosterone may act through the same mechanism to modulate secretion of LH, or once shut down, the GnRH pulse-generating system becomes refractory to stimulation by naloxone. In acutely castrated male rats, levels of LH were significantly increased above baseline throughout the period of naloxone treatment; this finding supports the hypothesis that the acute elevation in testosterone acting through mechanism independent of opioid is responsible for the transient response of LH to naloxone in the intact rat.     

Proopiomelanocortin (POMC) products in the central regulation of sympathetic and cardiovascular dynamics: studies on melanocortin and opioid interactions

The proopiomelanocortin (POMC)-derived peptides are important regulators in a number of central nervous system pathways especially as they relate to food intake as well as metabolic and autonomic responses. In this study, we investigated the sympathetic nervous and cardiovascular responses to intracerebroventricular (i.c.v.) administration of alpha melanocyte stimulating hormone (alphaMSH), beta-endorphin (beta-END) and adrenal corticotrophic hormone (ACTH) alone or in the presence of a melanocortin antagonist, or an opioid antagonist, in normal animals. The i.c.v. administration of alphaMSH and ACTH resulted in a significant increase in the lumbar sympathetic nerve activity (LSNA) that was accompanied by an increase in mean arterial pressure (MAP). On the other hand i.c.v. administration of beta-END decreased the LSNA and MAP. The pretreatment of animals with the melanocortin-4 (MC-4) receptor antagonist, agouti protein, significantly antagonized the response to alphaMSH and also, paradoxically, not only antagonized the response to beta-END but converted its inhibitory responses on both the LSNA and MAP to a sympathetic activated and pressor response. Pretreatment with the opioid antagonist, naloxone, significantly antagonized the sympathetic nervous and cardiovascular response to beta-END. It partially but significantly antagonized the MAP response to alphaMSH, but the sympathetic response was unaffected. Neither agouti protein nor naloxone altered the sympathetic nervous and cardiovascular response to ACTH. From these studies, we conclude that i.c.v. administration of alphaMSH and ACTH increases the LSNA and cardiovascular dynamics, whereas beta-END decreases it. And, the MC-4 receptor antagonist reverses the endorphin response and the opioid antagonist attenuates the alphaMSH response suggesting possible receptor or central neural pathway interactions between MC-4 and the opioid receptor mediated effects.     

Time course of plasma corticosterone, 18-hydroxycorticosterone and aldosterone concentrations following CRF administration in the rat. A phase of corticosterone inhibition

Synthetic ovine CRF (8 micrograms/rat) injected intravenously in nembutal anaesthetized rats increased not only plasma ACTH and corticosterone but also aldosterone and 18-hydroxycorticosterone concentrations. The maximum elevation occurred 30 min after oCRF administration. 2 h and 4 h after injection the hormone concentrations declined and after 6 h the corticosterone and 18-hydroxycorticosterone values were lower than the corresponding controls. At this time aldosterone remained slightly elevated and ACTH unchanged. 24 h after oCRF injection no difference between the control and oCRF treated animals were evident.     

Naloxone effect on ANP in trained and untrained rats

1. The effect of a dose of naloxone (1 mg·kg⁻¹ b.w.) on peripheral (plasma, atria) and central (hypothalamus, hypophysis) levels of atrial natriuretic peptide (ANP) was investigated in the rat.2. In control rats, an acute subcutaneous dose of naloxone produced no significant change in plasma ANP, but a decrease (NS) in atrial ANP concentration.3. In physically conditioned animals, naloxone produced a significant decrease in atrial ANP levels. Receptor sensitivity may thus be involved in this differential response.4. In hypothalamus and hypophysis, no effect on ANP concentrations was seen after a high dose of naloxone whether in control or in physically conditioned animals, suggesting peripheral and central ANP might be differently regulated, at least after chronic endurance physical training.     

Naloxone effect on ANP in trained and untrained rats.

1. The effect of a dose of naloxone (1 mg.kg-1 b.w.) on peripheral (plasma, atria) and central (hypothalamus, hypophysis) levels of atrial natriuretic peptide (ANP) was investigated in the rat. 2. In control rats, an acute subcutaneous dose of naloxone produced no significant change in plasma ANP, but a decrease (NS) in atrial ANP concentration. 3. In physically conditioned animals, naloxone produced a significant decrease in atrial ANP levels. Receptor sensitivity may thus be involved in this differential response. 4. In hypothalamus and hypophysis, no effect on ANP concentrations was seen after a high dose of naloxone whether in control or in physically conditioned animals, suggesting peripheral and central ANP might be differently regulated, at least after chronic endurance physical training.     

Aspects of opiate dependence in the myenteric plexus of the guinea-pig.

Myenteric plexus-longitudinal muscle strips prepared from tolerant/dependent guinea-pigs and continuously exposed to normorphine, display a contracture upon naloxone challenge. This phenomenon represents a sign of abstinence. Removal of the opiate by extensive washing resulted in the failure of naloxone to induce the abstinence sign, while the plexus still displayed considerable, although reduced, tolerance to morphine. Reexposure of withdrawn preparations to normorphine reinduced the ability to display the abstinence sign. Highly tolerant preparations exhibited a 30 fold increase in sensitivity to serotonin and prostaglandin E₁ when tested a few minutes after naloxone-precipitated withdrawal. Supersensitivity rapidly declined when normorphine was washed off the preparation, while reincubation of withdrawn tissues with the opiate resulted in reinduction of supersensitivity. The data confirms a close relationship between a state of tolerance and dependence (including display of the abstinence sign) and supersensitivity to putative neurotransmitters or neuromodulators, becoming evident following administration of naloxone.     

Fasting reduces the response of liver glycogen phosphorylase to physiological levels of epinephrine in rats

The effect of 24 h fasting on the response of rat liver glycogen phosphorylase activity to an i.v. bolus of 2.75, 5.50 or 22.00 nmol kg-1 of epinephrine was studied. Even the lowest dose increased activity of the a form of the enzyme in the liver of anesthetized, fed rats to approximately 70 - 80% of total enzyme activity two minutes after administration. Further increased epinephrine doses failed to potentiate the enzyme response significantly, but shortened the time necessary for attaining the response, and delayed the return of enzyme activity to control values. No activation of phosphorylase was demonstrable after 2.75 nmol kg-1 of the hormone injected to fasted rats, but after increasing the hormone dose to 5.50 nmol kg-1 the enzyme response was the same as in the corresponding fed group at 2 min, and after administering the highest dose both at 1 and 2 min. According to these results, an increased threshold to epinephrine should be added to the already described effects of fasting, i.e. decreased phosphorylase a and total enzyme activity and shortened response to catecholamines. The efficacy of the i.v. bolus of 5.50 nmol kg-1 of epinephrine in increasing plasma epinephrine level to the theoretical value of 27.5 pmol ml-1 was proven by measuring plasma epinephrine which increased during the first minute after hormone administration to 24.5 + 5.9 pmol ml-1, to decrease during an additional minute with a half life of cca 22.2 seconds.     

Naloxone-precipitated jumping in mice pretreated with acute injections of opioids.

Naloxone induced jumping was examined in mice pretreated with single dose of narcotic agonist (morphine, heroin, LAAM, methadone), mixed agonist-antagonist (pentazocine, cyclazocine, buprenorphine), or an enkephalin analog (D-met², pro⁵)-enkephalinamide. Acute sensitization to naloxone, as demonstrated by jumping, was observed after pretreatment with the narcotics, the enkephalin analog, and to a lesser degree after cyclazocine and pentazocine. Mice pretreated with buprenorphine did not jump in response to naloxone. This procedure may be of value in the rapid identification of drugs with a propensity to produce morphine-like physical dependence.     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

Inhibition of abstinence syndrome in opiate defendent mice by cyclo (His-Pro)

Intracerebral administration of cyclo (His-Pro), the postulated metabolite of thyroliberin (TRH, pGlu-His-Pro-NH₂) inhibited the naloxone induced withdrawal responses in morphine dependent mice. Mice were rendered dependent on morphine by the subcutaneous implantation of a pellet (containing 75 mg of morphine free base) for three days. Six hours after pellet removal, the naloxone ED₅₀ for the jumping response was found to be higher in mice injected with cyclo (His-Pro) compared with that of vehicle controls. Similarly, the hypothermic response observed following 50 μg/kg of naloxone given given 6 h after pellet removal or that seen with 100 μg/kg of naloxone given 24 h after pellet removal from morphine-dependent mice was inhibited by cyclo (His-Pro). Previously, we have shown similar results with TRH on the morphine abstinence syndrome. It appears, therefore, that cyclo (His-Pro) may be the active metabolite of TRH and analogs of cyclo (His-Pro) may be useful in blocking the symptoms of the opiate abstinence syndrome.     

ACTH and corticosterone response to naloxone and morphine in normal, hypophysectomized and dexamethasone-treated rats

The effect of opiate receptors blocker naloxone on ACTH and corticosterone secretion in normal, dexamethasone-treated and hypophysectomized rats was studied. A dose-related increase in plasma corticosterone level was found at 45 min after s.c. injection of naloxone in a dose range of 0.25-2.0 mg kg-1. The rise in plasma corticosterone was preceded by a slight increase in plasma ACTH. Acute morphine administration in a relatively low dose (6 mg kg-1 s.c.) induced a significant rise in both plasma ACTH and corticosterone levels. Dexamethasone treatment was followed by low basal corticosterone level, by total inhibition of the stress response and response to morphine injection, while the response to ACTH administration was normal. Under these circumstances as well as in rats 6 days after hypophysectomy, naloxone failed to increase plasma corticosterone levels. It is concluded that a direct stimulation of corticosteroid biosynthesis in adrenal cortex is not involved in the mechanism of naloxone-induced activation of pituitary-adrenocortical function.