Narcotic antagonists attenuate drinking induced by water deprivation in a primate

Pure narcotic antagonists such as naloxone and naltrexone have consistently been shown to attenuate drinking in the rat after periods of water deprivation. One objective of this study was to extend observations to a primate species, the squirrel monkey. Whereas naloxone and naltrexone have a greater relative affinity for opiate receptors preferentially binding morphine and other opiate alkaloids than for those with high affinity for the endogenous opioid peptides, diprenorphine, another pure opiate antagonist, binds with equally high affinity to both receptor subtypes. Therefore, a second objective was to determine the actions of diprenorphine on drinking in water-deprived rats and squirrel monkeys and to compare the effects of this drug to those of naloxone and naltrexone. All three narcotic antagonists suppressed water consumption of monkeys and rats deprived of water for 18 and 24 hr, respectively. Diprenorphine was the most potent compound tested in both species, producing significant reductions in water consumption of monkeys and rats at systemic doses as low as 0.01 and 0.1 mg/kg respectively. Moreover, diprenorphine was the longest acting of the three drugs in the monkey. These results demonstrate that the narcotic antagonists attenuate drinking in primates as well as in rodents and support the hypothesis that these drugs reduce water intake by interrupting the activity of endogenous opioid pathways mediating drinking behavior.     

Stereospecific aversive property of narcotic antagonists in morphine-free rats

If endogenous, morphine-like substances have physiological functions, narcotic antagonists should have effects $\text{in vivo}$ even in the absence of exogenous, narcotic agonists. This hypothesis was supported by studies of taste aversions conditioned with narcotic antagonists; rats drank smaller amounts of distinctively flavoured solutions when their consumption on previous occasions preceded injections of naloxone (1–10 mg/kg), naltrexone (3.2 mg/kg), Mr 1452 (10 mg/kg) or (-)-BC-2860 (10 mg/kg). Stereoisomers (i.e. Mr 1453, (+)-BC-2860) which were inactive as narcotic antagonists did not induce significant taste aversions. It was suggested that the consistency and stereospecificity of aversion with the antagonists gave some support to interpretations in terms of antagonist actions at receptors for endogenous opioids.     

Opiate binding to cerebroside sulfate: a model system for opiate-receptor interaction.

Cerebroside sulfate was shown to bind etorphine and levorphanol with high affinity. The relative potency of narcotic analgesics in preventing the binding of levorphanol to cerebroside sulfate correlated well with their reported analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Some preliminary animal data also imply the involvement of CS in opiate action We, therefore, propose that CS may serve as a useful “receptor” model for the study of opiate-receptor interaction $\text{in vitro}$.     

Effects of narcotic antagonists on fluid intake in the rat

The fluid intake (sweetened Enfamil) of rats that had been deprived of food and water for 24 hours was measured following the subcutaneous administration of eight narcotic antagonists and agonists and $\text{d}$-amphetamine. Drugs were tested over at least a 30-fold range of doses. Fluid intake was depressed by the highest dose of each drug, but only the narcotic antagonists naloxone, naltrexone and nalorphine produced dose-related decreases in fluid intake that were not associated with gross disturbances of behavior. The anorexigenic activity of these drugs in the rat does not appear to be related to the drugs' narcotic antagonist properties.     

The effects of narcotic analgesics and narcotic antagonists on ganglionic transmission in the rat.

The effects of narcotic analgesics, narcotic-antagonist analgesics and narcotic antagonists on ganglionic transmission in the superior cervical ganglia of the rat were studied $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$ administration of morphine, meperidine, methadone, pentazocine or naltrexone blocked ganglionic transmission. Levorphanol, cyclazocine, nalorphine and naloxone had no effect on ganglionic transmission in this procedure. $\text{In}$$\text{vitro}$ studies confirmed the $\text{in}$$\text{vivo}$ results with the exception of levorphanol, cyclazocine and nalorphine, which were also found to block ganglionic transmission $\text{in}$$\text{vitro}$. In both preparations, naloxone did not antagonize the effect of morphine, suggesting that the effects of morphine and the other opiates were nonspecific. Similar potency of $\text{d}$- and $\text{l}$-isomers of pentazocine and cyclazocine support this conclusion. The observation that naltrexone blocked ganglionic transmission, but the other pure narcotic antagonist, naloxone, was inactive is somewhat unique to this test procedure and possibly significant.     

Failure of tryptophan load-induced increases in brain serotonin to alter food intake in the rat

The effects on food consumption of 50 and 100 mg/kg $\text{1}$-tryptophan injections, versus control saline treatment, were compared in 24-hour food-deprived rats at two time points in the rats' daily light-dark cycle. No effect of the two tryptophan doses, relative to the saline treatment, on food intake was observed, although tryptophan loading significantly raised brain tryptophan, serotonin, and 5-hydroxyindoleacetic acid levels, in a dose-dependent manner, over baseline concentrations. Implications of these data for serotonergic modulation of food intake regulation are considered.     

Does dehydration contribute to retarded fetal growth in rats exposed to alcohol during gestation?

An earlier study showed that pregnant rats given ethanol in drinking water exhibited a significant degree of dehydration. The objective of the present study was to determine whether dehydration alone contributes to fetal growth retardation in alcohol treated rats. Female Sprague-Dawley rats were divided into 4 dietary groups. Group 1 (alcohol) received 20% ethanol in drinking water for four weeks prior to mating and 30% alcohol in drinking water throughout pregnancy and a stock diet $\text{ad libitum}$. Group 2 (pair-fed) was given an amount of food equal to that consumed by the alcohol group with the alcohol isocalorically substituted by corn starch. Water was available $\text{ad libitum}$. Group 3 (pair-water) was given an amount of food and water equal to that consumed by the alcohol animals. Group 4 ($\text{ad libitum}$) was given food and water $\text{ad libitum}$. On day 21 of gestation body weights of the alcohol exposed fetuses were significantly lower than those of the other three treatment groups. The difference in fetal body weights between the pair-fed and pair-water groups was not significant. Placentas were significantly heavier in the alcohol group than in the pair-fed and pair-water groups. Maternal plasma osmolality was significantly higher in the alcohol treated rats when compared to the pair-fed and $\text{ad libitum}$ controls but not the pair-water group. No significant differences were seen in fetal plasma osmolality among the four treatment groups. It is concluded that dehydration does not contribute significantly to retarded fetal growth in rats given alcohol in drinking water as the sole source of fluid prior to and during gestation.     

Effects of the intracerebroventricular injection of antinociceptive doses of acetylcholine on the stereospecific binding of 3H-dihydromorphine

The antinociceptive effects of intraventricularly administered acetylcholine (ACh) and its congeners have been demonstrated by previous investigators. The opiate receptor binding concept was used in this study to investigate possible correlations between ACh antinociception and its effects on opiate stereospecific binding. ACh $\text{in vitro}$ decreased the stereospecific binding of ³H-dihydromorphine in mouse brain homogenates. Such decrease was also observed in the brain homogenates of mice which had been treated with ACh intracerebroventricularly (i.v.t.). The decrease in the stereospecific binding of ³H-dihydromorphine induced by (i.v.t.) acetylcholine was inhibited by naloxone, atropine, cyclazocine and pentazocine. The $\text{d}$-isomers of cyclazocine and pentazocine were more potent than the $\text{l}$-isomers in antagonizing the inhibitory effects of i.v.t. acetylcholine upon the stereospecific binding of ³H-dihydromorphine to mouse brain homogenates. The same stereospecificity of these two narcotic analgesics in blocking acetylcholine had been previously observed in the tail-flick test. It is suggested that the antinociceptive effects of acetylcholine are related to the inhibition of opiate stereospecific binding, the mechanism of which is yet to be understood.     

Method of ethanol administration as a confounding factor in studies of fetal alcohol syndrome

Female Sprague-Dawley rats were fed a complete liquid diet containing either 5.5% ethanol (mean daily intake of about 9g of ethanol per kg body weight) or an isocaloric amount of dextrose (control group), with additional water available $\text{ad}$$\text{libitum}$. The diets were fed for four weeks prior to and throughout pregnancy. On day 20 of gestation cardiac output and blood flow to the placeta, heart, kidneys and uterus were measured and plasma osmolality and muscle dry weight were determined. No significant differences were seen between alcohol and control groups with respect to litter size, fetal weight, maternal cardiac output, blood flow to the placenta or other organs, plasma osmolality, or muscle dry weight. This contrasts with previous experiments in which a similar quantity of alcohol (as % calories) was offered in drinking water (equivalent to a mean daily ethanol intake of 10g/kg body weight). Under those conditions fetal weight was reduced, blood flow to the plascenta was reduced, and plasma osmolality and muscle dry weight were increased, indicating a moderate degree of dehydration. It is concluded that the effect of ethanol ingestion is influenced by the mode of administration of the ethanol. Dehydration may be a confounding factor in studies of animal models of fetal alcohol syndrome, although it is not possible to rule out a differential metabolic response to alcohol, depending on the mode of administration.     

Suppression by naloxone of water intake induced by deprevation and hypertonic saline in intact and hypophysectomized rats

Naloxone, an opiate antagonist, was administered to intact and hypophysectomized male rats following hypertonic saline pretreatment or 12 hr water deprivation. Water intake following hypertonic saline or water deprevation was reduced by 0.01 – 10 mg/kg of naloxone in a dose-related fashion in both intact and hypophysectomized rats. Water consumption induced by hypertonic saline administration appeared to be more susceptible to the suppressant effects of naloxone than did that evoked by water deprevation. These results demonstrate that naloxone reduces water intake in the rat following intracellular dehydration by hypertonic saline administration, as well as after general dehydration induced by water deprevation. Furthermore, the suppressant effects of naloxone on water intake do not appear to involve pituitary endorphins, although a possible involvement of antidiuretic hormone in these effects cannot be excluded.     

Naloxone antagonism of phenoxybenzamine antinociception in the mouse tail stimulation test.

Phenoxybenzamine was antinociceptive in the mouse tail electrical stimulation assay (ED50, 36.8 mg/kg) with a peak effect at $\text{2}\text{1}\text{2}$ hours after subcutaneous injection. Naloxone antagonized this antinociception action of phenoxybenzamine in a dose-related manner. Dose-ratio analysis of naloxone's antagonism of phenoxybenzamine antinociception gave a pA₂ value of 6.15, similar to that found for the benzomorphinan mixed agonist-antagonists. This is in agreement with the sodium response ratio found for phenoxybenzamine, 4.3, in $\text{in vitro}$ assays of phenoxybenzamine inhibition of ³H-naloxone binding to mouse brain homogenate (5). These findings suggest that phenoxybenzamine binds to the opiate receptor both $\text{in vivo}$ as well as $\text{in vitro}$ in a manner similar to the mixed agonist-antagonists.     

Synthesis of porcine leumorphin and some of its biological activities

The carboxy-terminal nonacosapeptide sequence of porcine preproenkephalin B contains the sequence of Leu-enkephalin at its amino terminus. The endogenous existence of this peptide, leumorphin, has not yet been proved. Synthesis of leumorphin was carried out by a solid-phase technique and the purity and structure of the synthetic peptide were confirmed. Synthetic porcine leumorphin exhibited a dose-dependent opiate effect (ED₅₀ 4.70 · 10^?9 M) on electrically stimulated contraction of the guinea pig ileum preparation. The potency was about 100 times as high as that of Leu-enkephalin. Leumorphin was less potent than dynorphin(1–13) (ED₅₀ 0.38 · 10^?9 M) but it was more active than $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{endorphin}$ (ED₅₀ 18 · 10^?9 M). The opiate activity was only partially reversed by naloxone. Intracisternal injection of synthetic leumorphin caused significant analgesia in mice (ED₅₀ 7.31 nmol/mouse). The potency was lower than that of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{endorphin}$ (ED₅₀ 0.60 nmol/mouse) but higher than that of dynorphin(1–13) (ED₅₀ 16.10 nmol/mouse). Intracisternally injected leumorphin did not produce such a violent behavioral effect as did dynorphin(1–13), and it exhibited a mild sedative effect. The data supports the concept that leumorphin is a new type of opioid peptide and that the synthetic preparation will be useful for further biological and immunological studies on this peptide.     

Mechanism of development of tolerance to injected morphine by guinea pig ileum.

Injection of a large dose of morphine into a guinea pig results in a block of electrically-induced contractions of the ileum $\text{in vitro}$. A similar dose is almost ineffective in guinea pigs given morphine chronically. The time course for development of this tolerance has been determined in guinea pigs injected twice daily with morphine 100 mg/kg and challenged on various days with 750 mg/kg of the drug. Animals similarly injected but not challenged served as controls. The inhibitory effect of the challenging dose on electrical stimulation of longitudinal muscle decreased with successive days of morphine administration; by the 10th day there was almost complete tolerance to the challenging dose. Sensitivity of the tissues of chronically morphinized unchallenged controls towards acetylcholine, serotonin, histamine and norepinephrine was essentially the same as that of naive animals. The potency of morphine $\text{in vitro}$ in blocking electrical stimulation was also unchanged by chronic morphine administration in the above manner. Thus tolerance to injected morphine cannot be explained by reduced affinity of the drug for the opiate receptor. Tissues of chronically morphinized animals gave a contracture with naloxone, the extent of the contracture increasing with time of drug administration. This naloxone effect is attributed to displacement of morphine from a new opiate receptor site induced during morphine administration. It is suggested that this new receptor is involved in tolerance to injected morphine as well as some aspects of the withdrawal syndrome.     

Placental blood flow in rats fed alcohol before and during gestation

Female Sprague-Dawley rats were either given 20% alcohol in drinking water and solid diet $\text{ad libitum}$ (alcohol group) or were pair-fed to the alcohol group (pair-fed group) or were given water and solid diet $\text{ad libitum (ad libitum}$ group) for four weeks. They were then mated and the alcohol group was changed to 30% alcohol in water. On day 20 of gestation each rat was injected with ⁵⁷Co-labeled microspheres into the left ventricle and radioactivity was determined in the placentas and kidneys. Cardiac output and blood flow to the placentas and kidneys was calculated. Fetuses and placentas were weighed, and the osmolality of the maternal plasma and water content of the muscle was determined. Cardiac output and blood flow to the kidneys did not differ among the three groups. Blood flow to the placenta, whether expressed as m1/min/g placenta or m1/min/placenta, or as % of cardiac output was significantly reduced in the alcohol group compared with the pair-fed and $\text{ad libitum}$ groups, which did not differ from one another. Fetuses were significantly lighter and placentas were significantly heavier in the alcohol group than in the other two groups. Plasma osmolality was increased and muscle water was decreased about 7% in the alcohol group, indicating a moderate degree of dehydration. It is concluded that chronic alcohol consumption leads to a redistribution of blood, with less blood supplying the placentas. This may contribute to the growth retardation seen in fetal alcohol syndrome.     

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.     

Complete,reversible, drug-specific tolerance to stimulation of locomotor activity by caffeine

The development of tolerance to caffeine-induced stimulation of locomotor activity was evaluated in rats maintained chronically on average daily doses of 160 mg/kg or more of caffeine by the method of scheduled access to drinking water containing the drug. Dose-response curves were determined for caffeine (6.25–100 mg/kg) and $\text{d}$-amphetamine (0.39–6.4 mg/kg) during chronic drug treatment. In addition, the caffeine curve was redetermined 2–3 weeks after removal of the drug from the drinking water. A control group that had scheduled access to drug-free tap water was also tested. Caffeine produced dose-related increases in the locomotor activity of the controls but failed to modify locomotor activity of the chronic caffeine group. In contrast, $\text{d}$-amphetamine increased locomotor activity of both groups comparably. Spontaneous locomotor of the chronic caffeine group was reduced significantly for 4 days after drug-free tap water was substituted for the caffeine solution. The return of spontaneous locomotor activity to baseline values was associated with restored sensitivity to caffeine-induced stimulation of locomotor activity. Thus, chronic administration of caffeine to rats results in the development of tolerance to caffeine-induced stimulation of locomotor activity that is virtually complete, pharmacologically specific, and fully reversible when drug treatment is stopped. Decreases in spontaneous locomotor activity after abrupt termination of chronic caffeine administration follow a time course consistent with a drug withdrawal syndrome.     

Effects of prolyl-leucyl-glycinamide and cyclo(leucyl-glycine) on morphine-induced antinociception and brain μ, δ and κ opiate receptors

The effects of prolyl-leucyl-glycinamide and cyclo (leucyl-glycine) on morphine-induced antinociception in mice and on $\text{in vitro}$ binding of ³H-ligands for opiate receptor subtypes (μ, δ and κ) the mouse brain homogenate were determined. Subcutaneous administration of either of the above peptides (1, 2, and 4 mg/kg) 10 min prior to the injection of morphine did not affect morphine-induced antinociception as evidenced by the identical ED₅₀ values of morphine in vehicle and peptide treated groups. The binding of ³H-dihydromorphine and ³H-naloxone ( μ receptors), ³HDAla²DLeu⁵-enkephalin (δ receptors), and ³H-ethylketocyclazocine (κ receptors) to opiate receptors in the mouse brain homogenate was also unaffected by both the peptides over a large concentration range. It is concluded that these peptides do not interact with brain opiate receptors.     

Acute inhibition of microsomal drug metabolism by propoxyphene in narcotic tolerant/dependent mice

Propoxyphene (Darvon) was compared to SKF 525-A, a prototypical inhibitor of hepatic microsomal mixed function oxidases, to assess propoxyphene's potential to inhibit drug metabolism in morphine tolerant/dependent mice. $\text{In vitro}$, both propoxyphene (K_i = 3.5 × 10^?5M) and SKF 525-A (K_i = 4.3 × 10^?6M) inhibited the activity of aminopyrine N-demethylase competitively in hepatic microsomes from tolerant/dependent animals. Propoxyphene and SKF 525-A were weaker, noncompetitive inhibitors of aniline hydroxylase activity. $\text{In vivo}$, equimolar doses (0.24 mmoles/kg, i.p.) of each compound inhibited both of the above monooxygenases in the 10,000$\text{g}$ supernatant fractions of livers from the tolerant/ dependent animals. Propoxyphene was 40–50% as potent an inhibitor of these activities as SKF 525-A. A dose (300 mg/kg) of propoxyphene napsylate, shown to prevent narcotic abstinence signs with no observable toxicity in withdrawing mice, significantly prolonged the blood levels of injected pentobarbital and tripled pentobarbital sleeping time in these animals. When administered at 300 mg/kg chronically, propoxyphene napsylate acted as an inducer of its own metabolism. Propoxyphene napsylate, then, given acutely to narcotic tolerant/dependent mice, is a potent inhibitor of microsomal drug metabolizing capacity. Given chronically, it enhances this capability.     

Evidence of possible opiate dependence during the behavioral depressant action of a single dose of morphine

Rats were trained to lever press for food pellets under a 20 response fixed ratio (FR 20) schedule of reinforcement. A single injection of 15 mg morphine SO₄/kg suppressed operant behavior for $\text{1}\text{1}\text{2}\text{–3}\text{1}\text{2}\text{hrs}$, after which time responding resumed at a reduced rate. When 0.25 mg naloxone HCl/kg was given during the recovery phase, the behavioral depressant effect of the narcotic was immediately reversed and operant performance returned to predrug rates. In contrast, when 0.5 mg naloxone/kg was given at this time, operant behavior was abolished for at least 1 hr. Naloxone, at these doses, did not affect responding in drug-naive subjects. These results suggest that a single, relatively low dose of morphine can induce transient dependence which is detectable for several hrs after drug administration, at a time when the acute pharmacological actions of morphine are still apparent.     

Differential effects of dopamine antagonists on prolactin secretion from cultured rat pituitary cells.

Various dopamine antagonists, including two novel non-neuroleptic drugs domperidone and halopemide, stimulated apomorphine-suppressed prolactin secretion from cultured rat pituitary cells. The potency of these drugs closely paralleled their rank-order in displacing $\text{in vitro}$ H³-haloperidol binding in rat striatum reported by others (10). Concentration-effect curves were parallel except those of pimozide and clopimozide which were biphasic : prolactin secretion was stimulated at low concentrations but depressed at concentrations above 25nM. When added alone, pimozide and clopimozide, but none of the other drugs tested, also depressed prolactin secretion. The present findings indicate that prolactin secretion from cultured pituitary cells may provide an $\text{in vitro}$ test system suitable to differentiate antagonists of dopamine receptors and possibly to distinguish pure from partial antagonists.