Neuroanatomical correlates of morphine dependence.

Naloxone hydrochloride, an opioid antagonist, was applied to several discrete brain regions of morphine-dependent rats to precipitate abstinence. Severe withdrawal signs were elicited after administration in the thalamus but not in neocortical, hippocampal, hypothalamic, or tegmental areas of the brain.

Morphine tolerance, physical dependence, and synthesis of brain 5-hydroxytryptamine.

Tolerance and physical dependence development to morphine in mice can be prevented by concomitant administration of cycloheximide. The fact that the rate of synthesis of brain 5-hydroxytryptamine (5HT) increases with tolerance to morphine suggests that the protein involved may be associated with 5HT synthesis. Inhibition of this synthesis with p-chlorophenylalanine markedly decreases tolerance and physical dependence development to morphine.

Effect of beta-endorphin on calcium uptake in the brain.

The uptake of 45Ca2+ by nerve-ending fractions from brains of mice was inhibited in vitro by 10(-9)M concentrations of beta-endorphin and in mice injected intraventricularly with 7 picomoles of beta-endorphin. That the effect was a specific opiate agonist response of beta-endorphin was demonstrated by use of the opiate antagonist, naloxone, which reversed the action. A role for beta-endorphin in the regulation of calcium flux and neurotransmitter release should be considered.

Modification of endorphin/enkephalin analgesia and stress-induced analgesia by divalent cations, a cation chelator and an ionophore.

The possibility that divalent cations may antagonize opiate peptide analgesia and stress-induced analgesia was examined. Intracerebroventricular injection of low doses of Ca2+, Mn2+ and Mg2+ antagonized beta-endorphin and methionine-enkephalin analgesia. Ba2+ and Cd2+ were without effect. The ionophore, A23187, significantly antagonized beta-endorphin analgesia and the effect was increased when a low dose of Ca2+ was injected at the same time as the ionophore. Ethylene glycol tetraacetic acid (but not ethylenediamine tetraacetic acid) significantly potentiated endorphin analgesia. Stress-induced analgesia, as determined by increased tail-flick latencies following intraperitoneal injection of acetic acid, was effectively antagonized by naloxone, Ca2+ and Mn2+. The frequency of writhing following acetic acid injection was increased by both naloxone and divalent metal ions, again suggesting antagonism of endogenous opiates. These results confirm previous findings indicating that divalent metal ions (and especially Ca2+) may be involved in the actions of opiates.

Contractile effect of morphine and related opioid alkaloids, beta-endorphin and methionine enkephalin on the isolated colon from Long Evans rats.

1 Morphine and related synthetic surrogates as well as beta-endorphin and methionine enkephalin caused a contractile response of the longitudinal musculature of the terminal colon of Long Evans rats.2 The muscular contraction caused by the narcotic analgesics exhibited stereospecificity, with levorphanol being about 50 times more potent than dextrorphan and (-)-methadone 4 times more potent than (+)-methadone. In addition, the rank order in potency of a homologous series of N-alkyl substituted norketobemidones demonstrated that the activity of these compounds in eliciting contractile responses corresponded to that for analgesic efficacy in the rat and also correlated to the ability of these derivatives to inhibit the muscular twitch evoked by electrical stimulation of the guinea-pig ileum.3 Naloxone blocked the contractile response of the opiates following competitive kinetics; the naloxone pA(2) values for morphine, etorphine, levorphanol and methadone were very close, in spite of the marked differences in potency of these agents.4 The contractile effect of morphine on the rat colon was abolished by incubation of the tissues with tetrodotoxin 2.0 x 10(-7) M or by decreasing the external Ca(2+) level 100 fold. Increasing the external Ca(2+) concentration caused an apparent non-competitive antagonism of the response to morphine.5 Pretreatment of the tissues with hexamethonium 8.3 x 10(-5) M caused a modest antagonism of the morphine effect while atropine 5.8 x 10(-7) M did not significantly modify the morphine contractile effect. In contrast, methysergide 10(-5) M caused a 10 fold increase in the morphine EC(50).6 Colons from rats rendered tolerant-dependent on morphine were markedly less sensitive to the contractile effects of morphine than those from placebo-treated controls. Tolerance to morphine was also accompanied by an increased sensitivity to the contractile effects of 5-hydroxytryptamine (5-HT).7 A marked increase in the spontaneous muscular activity of segments of the terminal colon of rats chronically treated with morphine was found to occur upon removal of the residual morphine in the tissues by repetitive washings. The spontaneous activity was arrested by applications of morphine, suggesting that physical dependence can be demonstrated in vitro in this particular preparation.8 It is concluded that the opiate-induced contractile response is mediated via stereospecific, naloxone-sensitive, opiate receptors and that the muscular response involves the activation of a 5-HT neurone in the nerve terminals of the colon.

Inhibition of Ca++-ATPase of rat erythrocyte membranes by k-opioid agonists.

The effect of various opioid agonists on Ca++, Mg++-ATPase activity of rat erythrocyte membranes was studied. The Ca++-stimulated component of this enzyme (Ca++-ATPase) showed properties similar to those of the Ca++-pumping ATPase of human erythrocyte membranes, that is, high affinity for Ca++, potentiation by calmodulin, and insensitiveness to Na+. Ethylketocyclazocine (EKC) dose-dependently inhibited this Ca++-ATPase activity at a concentration less than one nM without changing basal Mg++-ATPase activity and this action was reversed by the antagonist Win 44,441. Other opioid agonists mimicked this EKC effect and the rank order of potency was dynorphin (1-13) = greater than EKC greater than levorphanol = morphine = B-endorphin = dihydromorphine greater than leu-enkephalin greater than (D-Ala)2-(D-leu)5-enkephalin (DADL) = morphiceptin. It is concluded that rat erythrocyte membranes possess k-type opioid receptors through which the Ca++-pump is inhibited.

Opioid-like properties of seven dynorphin (1-10) analogs.

In order to disassociate the k action of dynorphin from its other actions, seven analogs were synthesized and evaluated for pharmacologic activity in comparison with dynorphin (1-13) and dynorphin amide (1-10). Dynorphin (1-10) was modified by protecting the terminal carboxy group, incorporating thioproline at position 10 and substituting methionine for leucine at position 5. All analogs exhibited the ability to inhibit electrically-induced twitches of the guinea pig ileum and mouse vas deferens in a manner that was dose dependent and naloxone reversible. The decapeptide terminating with a pyrrolidine group showed the highest potency in the ilea and mouse vas deferens. None of the analogs showed analgetic activity by the mouse tail flick test. Binding studies using mouse brain synaptosomes showed that all seven analogs can displace the binding of tritiated dihydromorphine (DHM), ethylketocyclazocine (EKC) and D-Ala-D-Leucine enkephalin (DADL). The alterations in chemical structure affected affinity of the analogs to the opiate receptor and their pharmacologic properties differently, suggesting that different opiate subtypes may be involved.

A high affinity Ca2+-ATPase in enriched nerve-ending plasma membranes.

A preparation of enriched synaptosomal plasma membrane (SPM) from mouse brain was found to contain a Mg2+-independent high affinity Ca2+-activated ATPase. The preparation readily accumulated Ca2+ at micromolar concentration from the medium in the presence or absence of Mg2+ and released previously accumulated calcium upon the addition of ATP. It is concluded that SPM Ca2+-ATPase may have a functional role in removal of cytosol Ca2+.

Calcium transport in and out of brain nerve endings in vitro--the role of synaptosomal plasma membrane Ca2+-ATPase in Ca2+-extrusion.

The effects of cellular cations and ATP on calcium transport in and out of the nerve endings (synaptosomes) of mice brain were studied. The synaptosomes accumulated 45Ca time-dependently in the absence of ATP or other additions for at least 10 min. When ATP was present, the overall 45Ca accumulation was decreased and was maximal at about 4 min, after which it started to decline. Studies on the effects of cations with or without ATP at 4 min revealed selective activities for different cations. Mg2+ inhibited 45Ca accumulation in the absence of ATP but increased 45Ca accumulation when ATP was present. Similarly, ATP increased 45Ca accumulation only when Mg2+ was present. Na+, on the other hand, inhibited 45Ca accumulation both in the presence and absence of ATP and/or Mg2+. K+ increased 45Ca accumulation in the presence of ATP with or without Mg2+; however, K+-stimulation was not noted in the presence of 100 mM Na+, and in fact, K+ became inhibitory. The ATP-stimulated 45Ca accumulation in the presence of Mg2+ peaked within 4-6 min and then declined, suggesting release of 45Ca. Compatible with this notion, in 45Ca-loaded synaptosomes, ATP evoked 45Ca release which was accompanied by the appearance of Pi in the medium. Although ATP-activated 45Ca-release can occur in the presence of Mg2+, Mg2+ is not required and, in fact, is inhibitory. Rapid release of 45Ca was also noted when 45Ca-loaded synaptosomes were incubated in the presence of Na+ without ATP. It is concluded that Mg2+, Na+, K+ and ATP each has a specific role in regulating Ca2+ permeability of the plasma membrane, calcium binding and calcium extrusion.

Effect of a calcium chelator on morphine tolerance development.

To assess the effects of calcium ion chelation on narcotic tolerance development, adult Sprague-Dawley rats were implanted with i.c.v. cannulae connected to osmotic minipumps for continuous infusions of saline or EGTA (2 mumol/24 h) during chronic morphinization by s.c. pellet implantation. Additional rats receiving the same morphine pellet treatment but without minipumps served as non-surgical controls. After 64 h the minipumps and pellets were removed and narcotic tolerance was assessed by the tail-flick technique. A positive analgetic response to an 8.50 mg/kg s.c. dose of morphine sulfate was noted in 6/12 no-minipump controls and 4/9 saline-infused controls. In placebo-treated rats caused no alteration of either baseline tail-flick latencies or in analgetic responsiveness to lower doses of morphine. It is concluded that the enhancement of morphine tolerance by EGTA (a calcium-specific chelator) results from a facilitation of certain adaptive changes of calcium ion disposition that are related to the neurochemical mechanisms of narcotic tolerance and dependence development.

Morphine tolerance and physical dependence: influence of cholinergic agonists and antagonists.

The effects of centrally acting agents which alter cholinergic activity were assessed in mice rendered tolerant to and dependent on morphine (M mice) and in naive mice (N mice). In both N and M mice, physostigmine potentiated morphine analgesia slightly, and this action was blocked by atropine and scopolamine. When administered 10 min before naloxone in dependent mice atropine enhanced precipitated withdrawal jumping; when given 30 min before naloxone, atropine produced an inhibition of the response. Physostigmine abd oxotremorine greatly inhibited the jumping response, while echothiophate had no effect. The inhibitory effect of physostigmine on naloxone precipitated withdrawal jumpimg was reversed by atropine and scopolamine but atropine did not alter morphine tolerance and dependence development. Brain acetylcholine (ACh) levels in both N and M mice were increased by physostigmine, the increase being greater in M mice. This increase was blocked by prior administration of atropine or scopolamine. When atropine was administered to M mice 10 min before sacrifice, brain AC-h levels decreased. However, when brain ACh levels were determined 30 min after atropine, no change was found. It was concluded that ACh does not play a major direct role in the development of tolerance and dependence, but that ACh is involved in the manifestations of acute morphine effects and in some of the withdrawal signs in the dependent state.

Dynorphin-(1-13) restores the potency of morphine on the tolerant guinea pig ileum.

Since it has been reported that dynorphin (1-13) enhances the analgetic effects of morphine in mice tolerant either to morphine or beta-endorphin and modulates opioid agonist binding in the tolerant state, experiments were initiated to determine whether comparable phenomenon could be obtained in vitro with dynorphin-(1-13) on the isolated longitudinal muscle of the guinea pig ileum. In the guinea pig ileum rendered tolerant to morphine dynorphin-(1-13) was found to cause a dose-dependent restoration in sensitivity to the inhibitory action of morphine. This phenomenon appeared specific for dynorphin-(1-13) since [Leu5]enkephalin, beta-endorphin, and ethylketocyclazocine did not change significantly the IC50 of morphine in the tolerant preparation. Furthermore, dynorphin-(1-13) attenuated the contraction elicited by naloxone in the morphine tolerant-dependent tissue. It is concluded, that dynorphin-(1-13) restores sensitivity to morphine in the tolerant-dependent state by a modulatory effect probably on the mu receptor that is distinct from its kappa action.

Methionine-enkephalin antagonism and endorphin potentiation of narcotic-induced analgesia.

Intracerebroventricular (i.c.v.) administration of subanalgesic doses of beta- and leucine-endorphin in mice 15 min before subcutaneous morphine injection, significantly enhanced the analgesic effects of the morphine as measured by the tail-flick assay. A similar effect was seen with levorphanol analgesia but not enhancement of leucine- or methionine-enkephalin analgesia by beta-endorphin was observed. The same doses of the endorphins did not affect the development of single-dose morphine tolerance and dependence. Leucine-enkephalin failed to affect morphine analgesia, tolerance or dependence development, while low doses of methionine-enkephalin administered i.c.v. were observed to have an antagonistic effect on morphine analgesia without affecting tolerance or dependence development. Neither endephalin had any effect on beta-endorphin analgesia.

Single-dose tolerance to antinociception, and physical dependence on beta-endorphin in mice.

beta-Endorphin (B-EN) injected intracerebroventricularly in mice produced a rapid onset, dose-dependent antinociceptive effect. The median analgesic dose (AD50) 30 min following administration was found to be 270 ng/mouse (3.7 nmoles/kg). B-EN produced an acute, single-dose tolerance which was characterized by its dose dependence and the time course of its development. Single-dose tolerance development was demonstrable with doses twice or more the AD50. Tolerance was maximal at about 12 h following the priming dose and disappeared within 48 h. Tolerance was accompanied by some degree of physical dependence as noted by signs of naloxone-precipitated withdrawal similar to those elicitable in the morphine-dependent state. Tolerance development to B-EN was blocked by the simultaneous administration of naloxone and also by pretreatment with 0.35 mg/kg actinomycin D or 30 mg/kg cycloheximide 30 min before B-EN. It appears that single-dose tolerance to B-EN was initiated by processes similar to those involved with tolerance resulting from chronic administration of morphine.

Potency of the N3im-methyl analog of TRH in the induction of shaking movements in the rat.

The relative potencies of TRH analogs in provoking a shaking response in rats were determined. Bilateral administration of 0.011-2.0 mug TRH analog into the periaqueductal-fourth ventricular spaces of the barbiturate-anesthetized rat showed that N3im-methyl TRH was approximately 10X more potent than TRH, whereas N1im-methyl TRH was approximately 10X less potent than TRH. These results indicate that the potencies of the TRH analogs in inducing shaking parallel their thyrotropin-releasing activities.

Alterations in the efficacy of naloxone induced by stress, cyclic adenosine monophosphate, and morphine tolerance.

Pretreatment of mice with a single injection of morphine or by chronic implantation of morphine pellets increased the ability of naloxone to antagonize the analgetic effects of morphine. However, this increased effectiveness of naloxone was also produced by pretreatment with diethylether, ACTH, corticosterone or dexamethasone. Thus, the increased potency of naloxone observed after pretreatment with narcotics may be due, at least in part, to those pretreatments on the pituitary--adrenal axis. In addition, in animals made highly tolerant and dependent by cAMP administration during morphine pellet implantation, the narcotic antagonist potency of naloxone was similar to that of untreated animals.

Effects of amino acids, especially taurine and gamma-aminobutyric acid (GABA), on analgesia and calcium depletion induced by morphine in mice.

The analgesic effect of morphine was antagonized in mice by intracerebroventricular pretreatment with taurine, gamma-aminobutyric acid (GABA) or glycine and was potentiated by ethylene glycol tetra-acetic acid (EGTA) but not altered by L-glutamate or L-aspartate. The potentiation of morphine analgesia by EGTA was reversed by a concentration of taurine that did not alter the tail-flick response. The selective depletion of 45Ca2+ from synaptic vesicles observed with morphine administration was significantly inhibited by taurine injection (1.2 mumol/brain, i.vt.) but was not altered by the same dose of GABA. Inhibition of ATP-dependent 45Ca2+ uptake in synaptosomes by morphine was also completely reversed by taurine (10(-2)M which by itself did not alter 45Ca2+ uptake. These results suggest that antagonism of morphine analgesia by taurine may be caused by blockade of the morphine-induced inhibition of both ATP-dependent synaptosomal 45Ca2+ uptake and changes in synaptic vesicular 45Ca2+ localization, while the antagonism by GABA was not associated with synaptosomal Ca2+.

Effect of 4-aminopyridine and verapamil on the inhibitory action of normorphine on the guinea-pig ileum.

The ability of normorphine to inhibit the muscular twitches elicited by electrical stimulation of the longitudinal muscle of the guinea-pig ileum was antagonized by 4-aminopyridine (4-AP). Incubation with 4-aminopyridine (4-AP) caused a marked increase of the normorphine IC50 and this effect was not modified by naloxone or by the Ca2+ channel blocker, verapamil. Although verapamil caused a significant decrease of the normorphine IC50 it also caused a potent reduction of the contractile effect of acetylcholine. The results suggest that 4-AP and verapamil act on different Ca2+ pools and that verapamil does not primarily affect the presynaptic Ca2+ pool involved in opiate action.

Behavioral interactions between naloxone and dopamine agonists.

A fixed-interval schedule of food presentation was used to study the effects of apomorphine, ET-495 and clonidine and interactions between these drugs and naloxone in the rat. The rate of responding under this schedule was decreased by low doses of apomorphine (0.03--0.3 mg/kg), ET-495 (0.3-1.0 mg/kg) and clonidine (0.03--0.3 mg/kg). Naloxone, at a dose (3 mg/kg) which did not itself alter responding, potentiated the rate decreasing effects of apomorphine and ET-495 but did not alter the effects of clonidine. It is suggested that the rate decreasing effects of low doses of apomorphine and ET-495 may be related to presynaptic inhibition of dopamine neurons and that under certain circumstances naloxone may also inhibit dopaminergic activity.

Potency of three opiate antagonists to reverse the inhibitory activity of dynorphin, enkephalins and opioid-like alkaloids on the guinea pig ileum.

To test the hypothesis that dynorphin is a K-opiate agonist acting on the myenteric plexus, the potency of two benzomorphan antagonists (Win 44, 441 and Mr 2266) to block the inhibitory action of dynorphin, enkephalins and opioid alkaloids was determined on the longitudinal muscle preparation of the guinea pig ileum. The effectiveness of these antagonists was compared to that of naloxone. Antagonistic potency was established by calculating the apparent antagonist dissociation constant, Ke, as derived from Schild plots. Win 44, 441 and Mr 2266 were about 7-8 times more potent than naloxone against dynorphin, dynorphin-(1-13) or ethylketocyclazocine. Although the Ke obtained with Win 44, 441 or Mr 2266 against dynorphin or ethylketocyclazocine were significantly lower than those of naloxone, the values obtained for these antagonists did not differ significantly in the case of each of these agonists. With respect to the antagonism of the enkephalins or normorphine, Win 44, 441 was the most potent antagonist. Its Ke value for the enkephalins was 2.5-3 times lower than those for dynorphin or ethylketocyclazocine and in comparison to naloxone, Win 44, 441 was about 5 times more potent. Although Mr 2266 was a potent antagonist of dynorphin, ethylketocyclazocine, the enkephalins or normorphine, it showed no selectivity of action. The fact that the 3 opiate antagonists evidenced similar Ke values for dynorphin and ethylketocyclazocine, but different ones for the enkephalins or normorphine supports the conclusion that dynorphin activates preferentially K- but not mu-opiate receptors in the myenteric plexus.