Nitrous oxide-induced hypothermia in the rat

Exposure of rats to high levels of nitrous oxide (N2O) in oxygen (O2) reduced body temperature in a concentration-related manner. The hypothermia was partly reversed by pretreatment with naloxone but not naltrexone. But in rats rendered tolerant to morphine by pellet implantation, exposure to 75% N2O/25% O2 evoked a marked hypothermia similar to that observed in morphine-naive animals. In another experiment, the hypothermic effect of chloral hydrate was also sensitive to antagonism by pretreatment with naloxone but not naltrexone. These observations lead us to suspect that N2O-induced hypothermia in rats is possibly not mediated by opiate receptors. The thermotropic activity of N2O may result from some non-opioid action of N2O. Its selective antagonism by naloxone (but not naltrexone) may be due to a unique non-opioid analeptic action of naloxone.     

Repeated administration of naltrexone produces tolerance to naloxene-induced hyperalgesia

Repeated administration (i.p.) of the narcotic antagonist naltrexone produced tolerance in mice to naloxone-induced hyperalgesia in the hot plate test. Chronic naltrexone treatment had no effect on pawlick latencies but produced significantly increased jump-reaction times in animals receiving an acute injection of saline. The findings are interpreted as indicating development of supersensitivity in endorphinergic systems mediating affective components in nociception.     

Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.     

Enhancement of pentobarbital effect by continuous administration of morphine in the mouse

These studies demonstrated that continuous morphine treatment from implantation of a 75 mg morphine pellet for 3 days potentiated pentobarbital narcosis and enhanced pentobarbital hypothermia. In the morphine implant mice, sleeping time after two different doses of pentobarbital was greater than 2.5 × the sleeping time in placebo pellet implant animals and also greater than sleeping time in animals treated acutely with morphine prior to pentobarbital. Moreover, in the morphine implant mice both the degree and duration of pentobarbital induced hypothermia were enhanced. The above findings were due to slower rate of metabolism of pentobarbital as evidenced by inhibition of hepatic N-demethylation, and higher levels of brain and serum pentobarbital in the morphine implant mice compared to both placebo and acute morphine mice.     

Ethanol-induced hypothermia. Cross tolerance with morphine

The development of tolerance to ethanol-induced hypothermia and hypnosis, and cross-tolerance with morphine was studied in mice and rats. Ethanol significantly decreased the body temperature in rats (3.0 and 3.2 g/kg) and in mice (3.5 and 4.0 g/kg). Chronic administration of ethanol resulted in the tolerance not only to ethanol hypothermia but also to hypothermic effects of morphine in examined animals. Implantation of morphine pellets caused the development of cross tolerance to ethanol-induced hypothermia in rats but not in mice. The hypnotic effect of ethanol was significantly shorter in chronic alcoholized rats but not in morphine-implanted rats. Neither chronic ethanol administration nor implantation of morphine pellets changed the duration of ethanol-induced hypnosis in mice. These results seem to support the hypothesis on the opiate-like mechanism of ethanol action.     

Effects of acute and chronic morphine and narcotic antagonists on brain energy metabolism

Morphine sulphate (4 mg/kg to 32 mg/kg) produced a dose-dependent decrease in brain malate as antinociception increased. Decreased brain malate persisted 72 hours after implantation of morphine pellets by which time mice had become tolerant to antinociception. This finding suggests that malate decrease, unlike changes of other metabolites in other studies, might not be simply a result of general metabolic changes. Malate change as well as antinociception was prevented by prior injection of naloxone (3.0 mg/kg) or naltrexone (0.6 mg/kg) in acute experiments. Malate decrease in pelleted mice was no longer present if withdrawal was produced by naloxone or naltrexone in mice implanted with morphine pellets for 72 hours. Brain P-creatine was elevated in all mice implanted with morphine pellets even after withdrawal, thus, apparently, representing a more generalized effect than malate change.     

Inhibition of pulsatile luteinizing hormone release by morphine microinjected into mesencephalic dorsal raphe

Blood samples were collected via jugular catheters from ovariectomized rats at 10-minute intervals for one hour before and two hours after microinjection of 0.5 μl of either saline vehicle or morphine sulfate (10 μg) into the dorsal raphe nucleus (DNR) or adjacent peri-aqueductal gray by means of chronically-implanted guide cannulae. LH was measured by radioimmunoassay and mean pre-injec post-injection values were compared for each rat (t test) as well as for each treatment group (paired t test). Neither saline in DRN nor morphine at other sites significantly altered circulating LH. A significant decrease in LH was observed following injection of morphine into DRN. This effect of morphine was prevented by pre treatment of the animals with the narcotic antagonist naltrexone (10 mg/kg i.v.), indicating the involvement of opiate receptors. These results indicate that DRN is one site at which systemically-administered morphine might act, and suggest the possibility of participation of this mechanism in modulation of LH release by endogenous opioids.     

Ethanol suppression of naloxone-induced withdrawal in morphine-dependent rats

The technique of morphine pellet implantation was used to produce physical dependence on morphine in male rats. The number of “wet dog” shakes occurring within a period of 30 minutes during naloxone-precipitated (1.0 mg/kg, s.c.) withdrawal in four-day morphine implanted rats was determined after either acute or chronic treatment with ethanol. An acute dose of ethanol administered prior to withdrawal had no significant effect on the withdrawal response whereas chronic administration of ethanol during the development of dependence on morphine significantly suppressed the naloxone-precipitated withdrawal response to 44–57 percent of the control response. Analysis of brain and plasma for morphine concentration four days following dependence development showed no significant differences between morphine controls and those animals treated with both morphine and ethanol. Pentobarbital, another central nervous system depressant, demonstrated no effect on the withdrawal response, whether administered acutely or chronically during the development of dependence.     

Differential effects of morphine on the rates of dopamine turnover in the neural and intermediate lobes of the rat pituitary gland

The acute administration of morphine to male rats decreased the rate of dopamine turnover in the median eminence and in the neural lobe of the pituitary, but was without effect in the intermediate lobe of the pituitary. Pretreatment with the opiate antagonist, naltrexone, reduced the effects of morphine. These results indicate that morphine, by acting on opiate receptors, inhibits the activity of tuberoinfundibular dopaminergic neurons that terminate in the median eminence and those tuberohypophysial dopaminergic neurons that terminate in the neural lobe of the pituitary.     

Influence of active and passive immunity on the disposition of dihydromorphine-H3

The effect of circulating antibodies on the plasma and brain concentration of dihydromorphine-³H was examined. Rabbits immunized with the morphine antigen 3-0-carboxymethyl morphine coupled to bovine serum albumin served as the source of the antibodies which were used for passive immunization of mice and rats. The 45 minute plasma concentration of the narcotic was increased 90–100 fold in passively immunized animals whereas the brain concentration decreased by at least 75 percent. Mice were also actively immunized with the morphine antigen. Compared to non-immunized mice, plasma levels of dihydromorphine-³H were increased in the immunized mice from 7–30 fold at all intervals measured for at least 4 days. The plasma half life was markedly slowed in immunized mice. The narcotic bound to the antibody in the plasma $\text{in vivo}$ could be displaced by the administration of morphine. The consequence of active immunization on brain narcotic content varied with the dose of the drug and time interval studied. We suggest the possibility that the antibodies may initially act to sequester the narcotic but with time as the narcotic is slowly released from the antibodies that they may also act as a circulating source of the drug. It is apparent that the presence of circulating antibodies can have marked effects on the disposition of narcotics.     

Apparent pA2 value of naltrexone is not changed in rats following continuous exposure to morphine or naloxone.

Chronic opioid antagonist administration increases opioid binding sites and potentiates behavioral responses to morphine. Conversely, chronic opioid agonist administration attenuates behavioral responses to morphine, though this is not necessarily accompanied by a parallel loss of binding sites. We examined the possibility that the in vivo affinity of the mu receptors might be altered as a consequence of the continuous administration of either naloxone or morphine. Rats were implanted sc with naloxone- or morphine-filled osmotic pumps; control animals were implanted with sham pumps. One week later, 24 hr after removing the osmotic pumps, cumulative dose-response curves for fentanyl analgesia were generated in the presence of 0.0, 0.03, 0.1, or 0.3 mg/kg naltrexone, using a tail-flick procedure. The analgesic ED50 (with 95% C. L.) of fentanyl in sham implanted animals, following saline pretreatment was 0.027 mg/kg (0.019, 0.039). The potency of fentanyl was decreased in rats infused with morphine, ED50 = 0.051 mg/kg (0.028, 0.093), and increased in rats that received naloxone, ED50 = 0.018 mg/kg (0.015, 0.022). The mean apparent pA2 value for naltrexone (with 95% C.L.) in the control group was 7.7 (7.5, 7.9). No differences were detected in animals that had received either naloxone or morphine for 7 days, pA2 = 7.8 (7.5, 8.1) and 7.4 (7.3, 7.6), respectively. Our results indicate that there is no change in the apparent affinity of the mu-receptor following continuous exposure to either an opioid agonist or antagonist, at a time when the analgesic potency of the agonist is decreased or increased, respectively.     

Narcotic antagonist activity of several metabolites of naloxone and naltrexone tested in morphine dependent mice (38558).

A rabbit liver enzyme system was used to produce the 6beta-OH reduced metabolites of naloxone and naltrexone. GC analysis indicated the presence of some 6alpha-OH metabolite in these samples. The narcotic antagonist activity of these 6beta-OH metabolite samples were compared to naloxone, naltrexone and standard 6alpha-OH naltrexone (EN-2260A) using the jumping response of morphine pellet implanted mice. For the naloxone series, the potencies were: Naloxone greater than EN 2265A greater than 6 beta-OH maloxone. For the naltrexone series: Naltrexone greater than EN 2260A greater than beta-OH naltrexone. The low potency of the reduced metabolites the rapid onset of action of the parent compounds militate against the formation of these metabolites contributing substantially to the overall narcotic antagonist action of the parent compounds.     

Effects of yohimbine and naltrexone in counteraction of the morphine straub tail and the amphetamine-potentiated morphine straub tail in mice

The alpha-adrenergic blocking agent, yohimbine, prevented the production of the morphine Straub tail reaction in mice, although on a mg dose basis it was only about $\text{1}\text{400}$ as potent as the narcotic antagonist naltrexone by subcutaneous injection. Likewise, yohimbine prevented the potentiation of the morphine Straub tail reaction by amphetamine, being about $\text{1}\text{170}$ as active as naltrexone. Preliminary studies with another alpha-adrenergic blocking agent, phentolamine, indicated that it also inhibited the production of the Straub tail reaction by morphine, although it appeared to be somewhat weaker than yohimbine in this respect. These results suggest the involvement of alpha-adrenergic mechanisms in the production of the morphine Straub tail reaction and in the potentiation of the morphine Straub tail reaction by amphetamine.     

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.     

Effects of morphine and beta-endorphin on basal and elevated plasma levels of alpha-MSH and vasopressin.

Morphine induced an increase of plasma α-MSH levels and a decrease of AVP levels after peripheral or intracerebroventricular administration. This increase of α-MSH levels and decrease of AVP levels after morphine treatment was observed in non-stimulated animals as well as in rats in which the hormone levels were elevated by water deprivation or by administration of hypertonic saline. These latter effects of morphine on plasma levels of α-MSH and AVP could be blocked by simultaneous administration of naltrexone.β-Endorphin also increased plasma α-MSH levels and lowered plasma AVP levels. From these effects only the increase of the plasma α-MSH level and not the decrease of plasma AVP could be blocked by naltrexone. Moreover PLG treatment was ineffective with respect to the endorphin-induced decrease in plasma AVP, but it partly blocked the increase of plasma α-MSH when this tripeptide was given in combination with β-endorphin.     

Lactic/glycolic acid polymers as narcotic antagonist delivery systems.

Lactic/glycolic acid polymers of several compositions were evaluated as the vehicle material for long term, controlled delivery of narcotic antagonists. L(+)-lactic, and glycolic acids-designated L(+), and G, respectively-were converted to polymers with weight ratios of 75 L(+)/25 G, 90 L(+)/10 G, and 100 L(+). Naltrexone base and naltrexone pamoate were incorporated into these polymers as a physical blend at several drug/polymer mass ratios. The mixtures were formed into small cylinders and spheres which were suitable for subcutaneous implantation by means of a trochar. $\text{In vitro}$ screening was carried out followed by $\text{in vivo}$ testing in mice. Radioactive assay and direct challenge with morphine using the tail-flick test were used to evaluate the drug release. The release rates approximated zero-order kinetics for most of the release period and the narcotic antagonist response to a challenge dose of morphine was maintained from one month to over six months depending on the formulation tested. Factors affecting narcotic antagonist delivery system design were polymer composition, narcotic antagonist solubility, drug loading level of the dosage form, use of a pure polymer coating around the drug/polymer matrix, and the surface area/unit volume of the dosage form.     

Can chronic pain be suppressed despite purported tolerance to narcotic analgesia?

Responsivity to acute nociceptive stimulation and the analgesic response to narcotic drugs was examined in rats exposed to an experimental model of chronic pain, i.e. $\text{Mycobacterium butyricum}$-induced adjuvant arthritis. The major findings are that (i) exposure to chronic pain alone causes hypo-algesia; this hypo-algesia can be attenuated by concurrent narcotics administration; (ii) chronic narcotics administration alone causes hyper-algesia; this hyper-algesia can be attenuated by concurrent exposure to chronic pain; (iii) the tolerance to narcotic analgesia which develops upon chronic narcotics administration in pain-free animals, need not occur in animals concurrently exposed to chronic pain. These findings support a recently proposed hypothesis on pain processing by the central nervous system, and may be suggestive of an effective treatment of chronic pain.     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.     

Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice

The present study used the endpoint of hypothermia to investigate opioid and neuropeptide FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important roles in thermoregulation, which suggested a link between opioid receptors and NPFF receptors in the production of hypothermia. Therefore, we designed a study to investigate the relationship between opioid and NPFF in control of thermoregulation in mice. The selective NPFF receptors antagonist RF9 (30nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol) after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third ventricle reduced the hypothermia induced by morphine (5nmol,) or nociceptin/orphanin FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol) nor NOP receptor antagonist [Nphe(1)]NC(1-13)NH(2) (7.5 nmol) reduced the hypothermia induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not modified. These results suggest that NPFF receptors activation is required for opioid to produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals.     

Quaternary narcotic antagonists' relative ability to prevent antinociception and gastrointestinal transit inhibition in morphine-treated rats as an index of peripheral selectivity

Single doses of naloxone (0.025 to 0.5 mg/kg) or of one of four quaternary narcotic antagonists (i.e. nalorphine allobromide, nalorphine methobromide, naloxone methobromide or naltrexone methobromide, 1 to 60 mg/kg) were given s.c. to rats before morphine, 5 mg/kg i.v. In the absence of antagonists morphine reduced G.I. transit of a charcoal meal to about 15% of drug-free controls and consistently delayed nociceptive reactions (55°C hot plate) in all animals. Doses of antagonists slightly reducing morphine antinociception (centrally effective = A) and restoring G.I. transit to about 50% of drug-free rats (peripherally effective = B) were estimated. The A:B ratio, indicating peripheral selectivity, was at least 8 for any of the quaternary antagonists given 10 min before morphine, but prolonging this interval may have resulted in a lower figure (i.e. less peripheral selectivity) because of reduced A and increased B. This was definitely so for naltrexone methobromide (A:B, > 60 at 10 min, about 1 at 80 min) and was not apparent for nalorphine methobromide according to available data, which for nalorphine allobromide and to a lesser extent for naloxone methobromide showed only an increase in B at intervals longer than 10 min. Both morphine-induced antinociception and inhibition of G.I. transit were reduced by naloxone at the lower doses tested and were fully prevented at the higher. These findings indicate that, unlike naloxone, the investigated quaternary narcotic antagonists are interesting prototype drugs for selective blockade of opiate receptors outside the CNS, although certain critical aspects, possibly biological N-dealkylation to the corresponding tertiary antagonists, condition peripheral selectivity.